Chemotherapeutic combinations of cationic antimicrobial peptides and chemotherapeutics

ABSTRACT

The present invention provides a compound, preferably a peptide, having the following characteristics: a) consisting of 9 amino acids in a linear arrangement; b) of those 9 amino acids, 5 are cationic and 4 have a lipophilic R group; c) at least one of said 9 amino acids is a non-genetically coded amino acid (e.g. a modified derivative of a genetically coded amino acid); and optionally d) the lipophilic and cationic residues are arranged such that there are no more than two of either type of residue adjacent to one another; and further optionally e) the molecule comprises two pairs of adjacent cationic amino acids and one or two pairs of adjacent lipophilic residues; for use in the treatment of a tumour by combined, sequential or separate administration with a cytotoxic chemotherapeutic agent that inhibits immune tolerance, wherein the chemotherapeutic agent is administered at a sub-cytotoxic dose. The present invention further provides pharmaceutical packs or compositions comprising these active agents and methods of treating a tumour comprising administration of these active agents.

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The present invention relates to peptides or peptide like molecules andparticularly to combined preparations of such peptides with a furtheragent, and their uses in therapy, in particular as anti-tumour agents.

The prevalence of cancer in human and animal populations and its role inmortality means there is a continuing need for new drugs which areeffective against tumours. Elimination of a tumour or a reduction in itssize or reducing the number of cancer cells circulating in the blood orlymph systems may be beneficial in a variety of ways; reducing pain ordiscomfort, preventing metastasis, facilitating operative intervention,prolonging life.

Genetic and epigenetic alterations that are characteristic of cancersresult in antigens that the immune system can recognise and use todifferentiate between tumour cells and their healthy equivalents. Inprinciple, this means that the immune system could be a powerful weaponin controlling tumours. However, the reality is that the immune systemusually does not provide a strong response to tumour cells. It is ofgreat therapeutic interest to manipulate and therefore harness theimmune system in the fight against cancer (Mellman et al. Nature 2011,vol. 480, 480-489).

Various attempts have been made to help the immune system to fighttumours. One early approach involved a general stimulation of the immunesystem, e.g. through the administration of bacteria (live or killed) toelicit a general immune response which would also be directed againstthe tumour. This is also called nonspecific immunity.

Recent approaches aimed at helping the immune system specifically torecognise tumour-specific antigens involve administration oftumour-specific antigens, typically combined with an adjuvant (asubstance which is known to cause or enhance an immune response) to thesubject. This approach requires the in vitro isolation and/or synthesisof antigens, which is costly and time consuming. Often not all thetumour-specific antigens have been identified, e.g. in breast cancer theknown antigens are found in 20-30% of the total tumours. The use oftumour-specific vaccines have therefore met with limited success.

There remains a strong need for alternative methods for treating tumoursand for alternative methods for inhibiting the growth or formation ofsecondary tumours.

‘Cancer Vaccine’ is a term used to describe therapeutic agents which aredesigned to stimulate the patient's immune system against tumourantigens and lead to an attack on tumour cells and improved patientoutcome. Despite the name, cancer vaccines are generally intended togenerate or enhance an immune response against an existing cancer,rather than to prevent disease. Again, unlike traditional vaccinesagainst infective agents, a cancer or tumour vaccine may not requireadministration of a tumour antigen, the administered product may utilisetumour antigens already present in the body as a result of tumourdevelopment and serve to modify the immune response to the existingtumour associated antigens (TAAs).

It is recognised that the usual lack of a powerful immune response toTAA is due to a combination of factors. T cells have a key role in theimmune response, which is initiated through antigen recognition by the Tcell receptor (TCR), and they coordinate a balance betweenco-stimulatory and inhibitory signals known as immune checkpoints(Pardoll, Nature 2012, vol. 12, 252-264). Inhibitory signals suppressthe immune system which is important for maintenance of self-toleranceand to protect tissues from damage when the immune system is respondingto pathogenic infection. However, immune suppression reduces what couldotherwise be a helpful response by the body to the development oftumours.

This T cell mediated balance of immune stimulation and suppression has,in recent years, led to the adoption of a principle of tumourimmunotherapy known as a ‘push-pull’ approach in which combinationtherapies could be used to simultaneously enhance the stimulatoryfactors (push) and reduce the inhibitory factors (pull). A helpfulanalogy is of a combination therapy which both presses on theaccelerator (push) and reduces the brakes (pull). (Berzofsky et al.Semin Oncol. 2012 June; 39(3) 348-57).

For example, cytokines, other stimulatory molecules such as CpG(stimulating dendritic cells), Toll-like receptor ligands and othermolecular adjuvants enhance the immune response. Co-stimulatoryinteractions involving T cells directly can be enhanced using agonisticantibodies to receptors including OX40, CD28, CD27 and CD137. These areall “push”-type approaches to cancer immunotherapy.

Complementary ‘pull’ therapies may block or deplete inhibitory cells ormolecules and include the use of antagonistic antibodies against whatare known as immune checkpoints. Immune checkpoints include CTLA-4 andPD-1 and antibodies against these are known in the art; ipilimumab wasthe first FDA-approved anti-immune checkpoint antibody licensed for thetreatment of metastatic melanoma and this blocks cytotoxic T-lymphocyteantigen 4 (CTLA-4) (Naidoo et al. British Journal of Cancer (2014) 111,2214-2219). There are other agents which would be considered classicchemotherapeutics which can reduce immune suppression at sub-cytotoxicdoses, these include cyclophosphamide and doxorubicin.

The present inventors have established that some peptides known to lysetumour cells through disturbing and permeabilizing the cell membrane,are also highly effective at attacking organelles such as mitochondriaand lysosomes and can cause lysis thereof. This may be achieved at lowconcentrations which do not cause direct lysis of the cell membranes,although loss of cell membrane integrity is seen eventually even onadministration of low doses. At higher doses, these molecules can causelysis of the cell membrane and then of the membranes of organelles.

The peptides of interest are a sub-set of the group of peptides commonlyknown as Cationic antimicrobial peptides (CAPs). These are positivelycharged amphipathic peptides and peptides of this type are found in manyspecies and form part of the innate immune system. The CAP Lactoferricin(LfcinB) is a 25 amino acid peptide which has been shown to have aneffect on mitochondria (Eliasen et al. Int. J. Cancer (2006) 119,493-450). It has now surprisingly been found that the much smallerpeptide LTX-315, a 9 amino acid peptide (of the type described in WO2010/060497), also targets the mitochondria. This was unexpected becausethis small peptide is much more fast acting (causing cell death after 30minutes of exposure) compared to LfcinB (which is most effective after24 hours of exposure) and the small peptide acts against a broaderspectrum of cell types, which suggests a direct effect on the plasmamembrane.

This disruption of the organelle membrane results in the release ofagents therefrom which have a potent immunostimulatory function, suchagents are generally known as DAMPs (Damage-associated molecular patternmolecules) and include ATP, Cytochrome C, mitochondrial CpG DNAsequences, mitochondrial formyl peptides, cathepsins (from lysosomes)and HMGB1 (from the nucleus). Lysis of organelles can also result inrelease of additional tumour-specific antigens (TAAs).

This ability to stimulate the immune response to tumours throughdisrupting mitochondrial and other organelle membranes makes thesepeptides highly suitable as “push” agents in combination “push-pull”immunotherapies designed to treat and protect against tumourdevelopment.

Thus, in a first aspect, the present invention provides:

A compound, preferably a peptide, having the following characteristics:

-   -   a) consisting of 9 amino acids in a linear arrangement;    -   b) of those 9 amino acids, 5 are cationic and 4 have a        lipophilic R group;    -   c) at least one of said 9 amino acids is a non-genetically coded        amino acid (e.g. a modified derivative of a genetically coded        amino acid); and optionally    -   d) the lipophilic and cationic residues are arranged such that        there are no more than two of either type of residue adjacent to        one another; and further optionally    -   e) the molecule comprises two pairs of adjacent cationic amino        acids and one or two pairs of adjacent lipophilic residues;        for use in the treatment of a tumour by combined, sequential or        separate administration with a cytotoxic chemotherapeutic agent        that inhibits immune tolerance, wherein the chemotherapeutic        agent is administered at a sub-cytotoxic dose.

The combination therapy proposed herein may, in certain advantageousembodiments, provide a synergistic effect.

The amino acid containing molecules defined above are convenientlyreferred to herein as the “peptidic compound of the invention”, whichexpression includes all of the peptides and peptidomimetics disclosedherein.

The cationic amino acids, which may be the same or different, arepreferably lysine or arginine but may be histidine or anynon-genetically coded amino acid carrying a positive charge at pH 7.0.Suitable non-genetically coded cationic amino acids include analogues oflysine, arginine and histidine such as homolysine, ornithine,diaminobutyric acid, diaminopimelic acid, diaminopropionic acid andhomoarginine as well as trimethylysine and trimethylornithine,4-aminopiperidine-4-carboxylic acid,4-amino-1-carbamimidoylpiperidine-4-carboxylic acid and4-guanidinophenylalanine.

Non-genetically coded amino acids include modified derivatives ofgenetically coded amino acids and naturally occurring amino acids otherthan the 20 standard amino acids of the genetic code. In this context, aD amino acid, while not strictly genetically coded, is not considered tobe a “non-genetically coded amino acid”, which should be structurally,not just stereospecifically, different from the 20 genetically coded Lamino acids. The molecules of the invention may have some or all of theamino acids present in the D form, preferably however all amino acidsare in the L form.

The lipophilic amino acids (i.e. amino acids with a lipophilic R group),which may be the same or different, all possess an R group with at least7, preferably at least 8 or 9, more preferably at least 10 non-hydrogenatoms. An amino acid with a lipophilic R group is referred to herein asa lipophilic amino acid. Typically the lipophilic R group has at leastone, preferably two cyclic groups, which may be fused or connected.

The lipophilic R group may contain hetero atoms such as 0, N or S buttypically there is no more than one heteroatom, preferably it isnitrogen. This R group will preferably have no more than 2 polar groups,more preferably none or one, most preferably none.

Tryptophan is a preferred lipophilic amino acid and the moleculespreferably comprise 1 to 3, more preferably 2 or 3, most preferably 3tryptophan residues. Further genetically coded lipophilic amino acidswhich may be incorporated are phenylalanine and tyrosine.

Preferably one of the lipophilic amino acids is a non-genetically codedamino acid. Most preferably the molecule consists of 3 genetically codedlipophilic amino acids, 5 genetically coded cationic amino acids and 1non-genetically coded lipophilic amino acid.

When the molecules include a non-genetically coded lipophilic amino acid(e.g. amino acid derivative), the R group of that amino acid preferablycontains no more than 35 non-hydrogen atoms, more preferably no morethan 30, most preferably no more than 25 non-hydrogen atoms.

Preferred non-genetically coded amino acids include:2-amino-3-(biphenyl-4-yl)propanoic acid (biphenylalanine),2-amino-3,3-diphenylpropanoic acid (diphenylalanine),2-amino-3-(anthracen-9-yl)propanoic acid,2-amino-3-(naphthalen-2-yl)propanoic acid,2-amino-3-(naphthalen-1-yl)propanoic acid,2-amino-3-[1,1:4′,1″-terphenyl-4-yl]-propionic acid,2-amino-3-(2,5,7-tri-tert-butyl-1H-indol-3-yl)propanoic acid,2-amino-3-[1,1′:3′,1″-terphenyl-4-yl]-propionic acid,2-amino-3-[1,1:2′,1″-terphenyl-4-yl]-propionic acid,2-amino-3-(4-naphthalen-2-yl-phenyl)-propionic acid,2-amino-3-(4′-butylbiphenyl-4-yl)propanoic acid,2-amino-3-[1,1′:3′,1″-terphenyl-5′-yl]-propionic acid and2-amino-3-(4-(2,2-diphenylethyl)phenyl)propanoic acid.

In a preferred embodiment the peptidic compounds of the invention haveone of formulae I to V listed below, in which C represents a cationicamino acid as defined above and L represents a lipophilic amino acid asdefined above. The amino acids being covalently linked, preferably bypeptide bonds resulting in a true peptide or by other linkages resultingin a peptidomimetic, peptides being preferred. The free amino or carboxyterminals of these molecules may be modified, the carboxy terminus ispreferably modified to remove the negative charge, most preferably thecarboxy terminus is amidated, this amide group may be substituted.

(I) (SEQ ID NO: 1) CCLLCCLLC (II) (SEQ ID NO: 2) LCCLLCCLC (III)(SEQ ID NO: 3) CLLCCLLCC (IV) (SEQ ID NO: 4) CCLLCLLCC (V)(SEQ ID NO: 5) CLCCLLCCL

A peptidomimetic is typically characterised by retaining the polarity,three dimensional size and functionality (bioactivity) of its peptideequivalent but wherein the peptide bonds have been replaced, often bymore stable linkages. By ‘stable’ is meant more resistant to enzymaticdegradation by hydrolytic enzymes. Generally, the bond which replacesthe amide bond (amide bond surrogate) conserves many of the propertiesof the amide bond, e.g. conformation, steric bulk, electrostaticcharacter, possibility for hydrogen bonding etc. Chapter 14 of “DrugDesign and Development”, Krogsgaard, Larsen, Liljefors and Madsen (Eds)1996, Horwood Acad. Pub provides a general discussion of techniques forthe design and synthesis of peptidomimetics. In the present case, wherethe molecule is reacting with a membrane rather than the specific activesite of an enzyme, some of the problems described of exactly mimickingaffinity and efficacy or substrate function are not relevant and apeptidomimetic can be readily prepared based on a given peptidestructure or a motif of required functional groups. Suitable amide bondsurrogates include the following groups: N-alkylation (Schmidt, R. etal., Int. J. Peptide Protein Res., 1995, 46, 47), retro-inverse amide(Chorev, M and Goodman, M., Acc. Chem. Res, 1993, 26, 266), thioamide(Sherman D. B. and Spatola, A. F. J. Am. Chem. Soc., 1990, 112, 433),thioester, phosphonate, ketomethylene (Hoffman, R. V. and Kim, H. O. J.Org. Chem., 1995, 60, 5107), hydroxymethylene, fluorovinyl(Allmendinger, T. et al., Tetrahydron Lett., 1990, 31, 7297), vinyl,methyleneamino (Sasaki, Y and Abe, J. Chem. Pharm. Bull. 1997 45, 13),methylenethio (Spatola, A. F., Methods Neurosci, 1993, 13, 19), alkane(Lavielle, S. et. al., Int. J. Peptide Protein Res., 1993, 42, 270) andsulfonamido (Luisi, G. et al. Tetrahedron Lett. 1993, 34, 2391).

The peptidomimetic compounds may have 9 identifiable sub-units which areapproximately equivalent in size and function to the 9 cationic andlipophilic amino acids. The term ‘amino acid’ may thus conveniently beused herein to refer to the equivalent sub-units of a peptidomimeticcompound. Moreover, peptidomimetics may have groups equivalent to the Rgroups of amino acids and discussion herein of suitable R groups and ofN and C terminal modifying groups applies, mutatis mutandis, topeptidomimetic compounds.

As is discussed in “Drug Design and Development”, Krogsgaard et al.,1996, as well as replacement of amide bonds, peptidomimetics may involvethe replacement of larger structural moieties with di- ortripeptidomimetic structures and in this case, mimetic moietiesinvolving the peptide bond, such as azole-derived mimetics may be usedas dipeptide replacements. Peptidomimetics and thus peptidomimeticbackbones wherein just the amide bonds have been replaced as discussedabove are, however, preferred.

Suitable peptidomimetics include reduced peptides where the amide bondhas been reduced to a methylene amine by treatment with a reducing agente.g. borane or a hydride reagent such as lithium aluminium-hydride. Sucha reduction has the added advantage of increasing the overallcationicity of the molecule.

Other peptidomimetics include peptoids formed, for example, by thestepwise synthesis of amide-functionalised polyglycines. Somepeptidomimetic backbones will be readily available from their peptideprecursors, such as peptides which have been permethylated, suitablemethods are described by Ostresh, J. M. et al. in Proc. Natl. Acad. Sci.USA (1994) 91, 11138-11142. Strongly basic conditions will favourN-methylation over O-methylation and result in methylation of some orall of the nitrogen atoms in the peptide bonds and the N-terminalnitrogen.

Preferred peptidomimetic backbones include polyesters, polyamines andderivatives thereof as well as substituted alkanes and alkenes. Thepeptidomimetics will preferably have N and C termini which may bemodified as discussed herein.

β and γ amino acids as well as a amino acids are included within theterm ‘amino acids’, as are N-substituted glycines. The peptidiccompounds of the invention include beta peptides and depsipeptides.

As discussed above, the peptidic compounds of the invention incorporateat least one, and preferably one, non-genetically coded amino acid. Whenthis residue is denoted L′, preferred compounds are represented by thefollowing formulae:

(I′) (SEQ ID NO: 6) CCL′LCCLLC (I′′) (SEQ ID NO: 7) CCLLCCLL′C (I′′)(SEQ ID NO: 8) CCLL′CCLLC (II′) (SEQ ID NO: 9) LCCLL′CCLC

Particularly preferred are compounds (preferably peptides) of formula Iand II, and of these, compounds (preferably peptides) of formula I″ areespecially preferred.

The following peptides as presented in Table 1 are most preferred.

TABLE 1 SEQ ID Name NO Sequence LTX-301 10 Dip-K-K-W-W-K-K-W-K-NH₂LTX-302 11 W-K-K-W-Dip-K-K-W-K-NH₂ LTX-303 12 W-K-K-W-W-K-K-Dip-K-NH₂LTX-304 13 Bip-K-K-W-W-K-K-W-K-NH₂ LTX-305 14 W-K-K-Bip-W-K-K-W-K-NH₂LTX-306 15 w-k-k-w-dip-k-k-w-k-NH₂ LTX-307 16 K-K-W-Dip-K-K-W-W-K-NH₂LTX-308 17 k-k-W-Dip-k-k-W-W-k-NH₂ LTX-309 18 K-K-W-Dip-K-K-W-Dip-K-NH₂LTX-310 19 K-K-W-Bip-K-K-W-W-K-NH₂ LTX-312 20 K-Bip-K-K-W-W-K-K-W-NH₂LTX-313 21 K-K-Bip-W-K-K-W-W-K-NH₂ LTX-314 22 K-K-W-W-K-K-Dip-W-K-NH₂LTX-315 23 K-K-W-W-K-K-W-Dip-K-NH₂ LTX-316 24 K-W-Dip-K-K-W-W-K-K-NH₂LTX-317 25 K-K-W-W-K-W-Dip-K-K-NH₂ LTX-318 26Orn-Orn-W-Dip-Orn-Orn-W-W-Orn-NH₂ LTX-319 27Dap-Dap-W-Dip-Dap-Dap-W-W-Dap-NH₂ LTX-320 28 R-R-W-Dip-R-R-W-W-R-NH₂LTX-321 29 K-W-W-K-K-Dip-W-K-K-NH₂ LTX-323 30 K-Dip-K-K-W-W-K-K-W-NH₂LTX-324 31 K-K-Dip-W-K-K-W-W-K-NH₂ LTX-325 32 k-w-w-k-k-dip-w-k-k-NH₂LTX-326 33 R-R-Bip-W-R-R-W-W-R-NH₂ LTX-327 34 R-R-Dip-W-R-R-W-W-R-NH₂LTX-329 35 k-k-bip-w-k-k-w-w-k-NH₂ LTX-331 36 k-k-Bip-w-k-k-w-w-k-NH₂LTX-332 37 K-K-bip-W-K-K-W-W-K-NH₂ LTX-333 38Dab-Dab-W-Dip-Dab-Dab-W-W-Dab-NH₂ LTX-334 39 K-K-W-1-Nal-K-K-W-W-K-NH₂LTX-335 40 K-K-W-2-Nal-K-K-W-W-K-NH₂ LTX-336 41 K-K-W-Ath-K-K-W-W-K-NH₂LTX-338 42 K-K-W-Phe(4-4′Bip)-K-K-W-W-K-NH₂

In which:

-   -   the standard single letter code is used for the genetically        coded amino acids    -   lower case denotes D amino acids    -   Dip is diphenylalanine    -   Bip is biphenylalanine    -   Orn is ornithine    -   Dap is 2,3-diaminopropionic acid    -   Dab is 2,4-diaminobutyric acid    -   1-NaI is 1-naphthylalanine    -   2-NaI is 2-naphthylalanine    -   Ath is 2-amino-3-(anthracen-9-yl)propanoic acid    -   Phe(4,4′Bip) is 2-amino-3-[1,1′:4′,1″-terphenyl-4-yl]propionic        acid

Compound LTX-315 is most preferred.

All of the molecules described herein may be in salt, ester or amideform.

The molecules are preferably peptides and preferably have a modified,particularly an amidated, C-terminus. Amidated peptides may themselvesbe in salt form and acetate forms are preferred. Suitablephysiologically acceptable salts are well known in the art and includesalts of inorganic or organic acids, and include trifluoracetate as wellas acetate and salts formed with HCl.

The peptidic compounds described herein are amphipathic in nature, their2° structure, which may or may not tend towards the formation of anα-helix, provides an amphipathic molecule in physiological conditions.

The combination therapies defined herein are for the treatment oftumours, in particular solid tumours and thus for the treatment ofcancer.

The peptidic compounds of the invention destabilise and/or permeabilisethe membranes of tumour cell organelles, e.g. mitochondria, the nucleusor lysomome, in particular the mitochondria.

By ‘destabilising’ is meant a perturbation of the normal lipid bi-layerconfiguration including but not limited to membrane thinning, increasedmembrane permeability to water, ions or metabolites etc.

A “cytotoxic chemotherapeutic agent” is an agent that which, at amaximum tolerated dose (MTD), is effective at killing cells that dividerapidly and thus may be used as an anticancer therapy. These drugstypically act by interfering with DNA synthesis or producing chemicaldamage to DNA. Such agents will not be effective in all patients but areprescribed for this ability to kill rapidly dividing cancer cells.Cytotoxicity and chemotherapy are terms that are well known in the fieldof oncology. The term does not cover agents that are discriminatelycytotoxic (agents that are able to kill cancer cells specifically) suchas monoclonal antibodies. The skilled person is aware of the cytotoxicchemotherapeutic agents used in the art and is also able to readilydetermine whether a putative agent has cytotoxic chemotherapeuticproperties. The skilled person may for example carry out ananti-proliferative assay, such as an MTS assay described in Example 1,against rapidly dividing cells. As discussed further below, these agentsare not used for their ability to kill rapidly dividing tumour cells andthe dose employed is termed “sub-cytotoxic”, i.e. less than cytotoxic.

Some cytotoxic chemotherapeutic agents inhibit immune tolerance (i.e.reduce the mechanisms of immune suppression). As described in Zheng etal (2015) Cell. Immunol., 294, pp 54-9, specific immune cells, effectormolecules and signal pathways can act in a process termed immunetolerance, resulting in tumour growth rather than an inhibition intumour growth. Regulatory T cells (Treg), myeloid-derived suppressorcells (MDSC) and tumour-associated macrophages (TAM) have key roles inwhat may be a cancer-induced immune tolerance. Cytotoxicchemotherapeutic agents have been reported to disrupt this immunetolerance leading to an immune response against the tumour cells. Agentsthat have been shown to stimulate immune activation include doxorubicin(Alizadeh et al. Cancer Res. (2014) 74(1); 104-118), paclitaxel (Michelset al. J. Immunotoxicol. (2012) 9(3); 292-300), cyclophosphamide(Heylmann et al. PLOS one, (2013) Vol. 8, Issue 12, e83384), gemcitabineand 5-fluorouracil; the chemotherapeutic agent of the invention ispreferably one listed here, more preferably cyclophosphamide ordoxorubicin.

Standard cytotoxic chemotherapeutic agents have many unwanted sideeffects. However, it has been observed (Heylmann, Alizadeh and Michelssupra) that some chemotherapeutic agents at sub-cytotoxic concentrationscan effectively facilitate immune activation with little toxicity andwith few of the challenging side effects of conventional chemotherapy.At these sub-cytotoxic concentrations the agents can reduce immunetolerance and thus enhance anti-cancer immunity, for example bydepleting highly proliferative immunosuppressive cells while sparingless proliferative lymphocytes with a protective function.Immunosuppressive cells such as regulatory T cells and MDSCs are targetsfor these agents, as are cytokines, which may all serve to preventcytotoxic T lymphocytes and natural killer cells from killing tumourcells.

The person skilled in the art would readily be able to determine whethera cytotoxic chemotherapeutic agent inhibited immune tolerance, e.g.through monitoring for immune tolerance changes such as dendritic cellmaturation, an increase in the expression of auxiliary receptors in Tcells and a decrease in immunosuppressive cells after administration ofthe agent in vivo, e.g. in an animal model or clinical study. Suchmonitoring may be carried out, for example, either usingimmunhistochemistry on isolated tumor tissue or through usingflowcytometry on single cells suspensions isolated from tumor tissue.The degree of inhibition will be therapeutically significant and may beat least 10% or 20% preferably at least 30% or 40%, more preferably atleast 50% of 60% as compared to a suitable control.

A “sub-cytotoxic dose” of the chemotherapeutic agent is one that is lessthan the dose usually prescribed to directly kill cancer cells, e.g.less than the MTD for standard chemotherapy. For cytotoxicchemotherapeutic agents used clinically, these cytotoxic doses arecommonly known in the art and may be set out in any regulatoryapprovals. For example a common dose of cyclophosphamide used inanti-cancer therapy is 40 to 50 mg/kg divided over 2 to 5 days, whichequates to 8 mg/kg/day at the lower end of the dosage range. Therefore,a non-cytotoxic dose of cyclophosphamide would be less than 8 mg/kg/dayin humans, typically less than 6 or 4 mg/kg/day.

Preferred sub-cytotoxic doses are between 1% (e.g. 5%) and 90% of thedose typically used for direct anti-cancer therapy. The dose may be, forexample less than 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% of thestandard cytotoxic dose. Preferably the dose is between 5% and 50%, morepreferably between 10% and 40%. Suitable sub-cytotoxic doses, oftenreferred to simply as ‘low dose’; are known and described in the art,e.g. in the art cited herein.

The non-cytotoxic dose may be still be toxic to non-cancerousrapidly-dividing cells, and such a dose may still cause adverse drugreactions (ADRs) in the subject. For example, a non-cytotoxic dose maystill adversely affect haematopoiesis in a subject.

For cytotoxic chemotherapeutic agents that are not commonly usedclinically, a sub-cytotoxic dose may be determined in vivo throughgradually increasing the titration of a dose and regular monitoring foreffects on cytotoxicity.

The sub-cytotoxic dose may be administered as a single dose or asmultiple doses. The non-cytotoxic dose may be administered in blocks,such as once or twice a day for 3-7 days, followed by a break of 3-7days, followed by a further course of administration. Even if multipledoses are administered, the impact on the body and the classification ofthe dosage regimen which would be understood by the clinician is still“sub-cytotoxic”. Thus, alternatively viewed, the uses and methods of theinvention employ a cytotoxic chemotherapeutic agent as defined hereinadministered according to a sub-cytotoxic dosage regimen.

Preferably the sub-cytotoxic dose is a metronomic dose (metronomicdosing is typically intended to be anti-angiogenic). A metronomic doseis a non-cytotoxic dose administered on a daily basis or every other dayfor a prolonged period of time, for example at least 2, 3, 4 or 6 weeks.Typically 6 to 45 doses, e.g. 6 to 20 doses.

The invention provides methods of treating a tumour and a method oftreating tumour cells. The combination therapy should be effective tokill all or a proportion of the target tumour cells or to prevent orreduce their rate of multiplication, or to inhibit metastasis orotherwise to lessen the harmful effect of the tumour on the patient. Theclinician or patient should observe improvement in one or more of theparameters or symptoms associated with the tumour. Administration mayalso be prophylactic and this is encompassed by the term “treatment”.The patient will typically be a human patient but non-human animals,such as domestic or livestock animals may also be treated.

Cancer targets include sarcomas, lymphomas, leukemias, neuroblastomasand glioblastomas (e.g. from the brain), carcinomas and adenocarcinomas(particularly from the breast, colon, kidney, liver (e.g. hepatocellularcarcinoma), lung, ovary, pancreas, prostate and skin) and melanomas.Breast, head and neck cancers are preferred targets. Melanoma, sarcomaand lymphoma are preferred tumour types for treatment. Tumours fortreatment are typically solid tumours and may be metastatic lesions thatare accessible for transdermal injection.

The peptides may be synthesised in any convenient way. Generally thereactive groups present (for example amino, thiol and/or carboxyl) willbe protected during overall synthesis. The final step in the synthesiswill thus be the deprotection of a protected derivative of theinvention. In building up the peptide, one can in principle start eitherat the C-terminal or the N-terminal although the C-terminal startingprocedure is preferred. Methods of peptide synthesis are well known inthe art but for the present invention it may be particularly convenientto carry out the synthesis on a solid phase support, such supports beingwell known in the art. A wide choice of protecting groups for aminoacids which are used in the synthesis of peptides are known.

References and techniques for synthesising peptidomimetic compounds andthe other bioactive molecules of the invention are described herein andare well known in the art.

While it is possible for the peptidic compounds (including salts, estersor amides thereof) to be administered as pure compounds, it ispreferable to present them as pharmaceutical formulations, i.e.incorporating one or more pharmaceutically acceptable diluents, carriersor excipients.

The active agents according to the invention may be presented, forexample, in a form suitable for oral, topical, nasal, parenteral,intravenal, intratumoral, rectal or regional (e.g. isolated limbperfusion) administration. Unless otherwise stated, administration istypically by a parenteral route, preferably by injection subcutaneously,intramuscularly, intracapsularly, intraspinaly, intrapentonealy,intratumouraly, transdermally or intravenously. For the peptidiccompound, administration is preferably intratumoural. Particularlypreferred are intratumoural injections of the peptidic compound of theinvention once a day for several consecutive days, e.g. 2, 3, 4, 5, 6 or7 days, preferably on 2-4 consecutive days, or at 2, 3, 4, 5, 6 or 7daily intervals.

The peptidic compound is preferably administered with or after thechemotherapeutic agent and there are preferably multiple administrationsof the peptidic compound, preferably 2-4, e.g. 3 administrations.

The active compounds defined herein may be presented in the conventionalpharmacological forms of administration, such as tablets, coatedtablets, nasal sprays, solutions, emulsions, liposomes, powders,capsules or sustained release forms. Conventional pharmaceuticalexcipients as well as the usual methods of production may be employedfor the preparation of these forms. Simple solutions are preferred.

Organ specific carrier systems may also be used.

Injection solutions may, for example, be produced in the conventionalmanner, such as by the addition of preservation agents, such asp-hydroxybenzoates, or stabilizers, such as EDTA. The solutions are thenfilled into injection vials or ampoules.

Preferred formulations are those in which the molecules are in saline.Such formulations being suitable for use in preferred methods ofadministration, especially local administration, i.e. intratumoural,e.g. by injection.

Unless otherwise stated, dosage units containing the peptidic moleculespreferably contain 0.1-10 mg, for example 1-5 mg. The formulation mayadditionally comprise further active ingredients, including othercytotoxic agents such as other anti-tumour peptides. Other activeingredients may include different types of cytokines e.g. IFN-γ, TNF,CSF and growth factors, immunomodulators, chemotherapeutics e.g.cisplatin or antibodies or cancer vaccines.

Also provided according to the present invention is the use of apeptidic compound as defined above in the manufacture of a medicamentfor the treatment of a tumour, wherein said peptidic compound isco-administered with a chemotherapeutic agent as defined above, at asub-cytotoxic dose.

Preferably, the medicament is for the treatment of multidrug resistant(MDR) tumours.

Also provided according to the present invention is a pharmaceuticalpack or composition comprising:

-   -   (i) a peptidic compound as defined herein; and    -   (ii) a chemotherapeutic agent as described herein, at a        sub-cytotoxic dose.

With pharmaceutical packs, the components can be for administrationseparately. The pharmaceutical pack can of course also compriseinstructions for administration. The pack and composition are for use inthe treatment of a tumour.

Also provided according to the present invention is a method oftreatment of a tumour, comprising the step of administering a peptidiccompound as defined herein and a chemotherapeutic agent as describedherein at a sub-cytotoxic dose, together in pharmaceutically effectiveamounts, to a patient in need of same.

As discussed above, the peptidic compounds of the invention are able todestabilise mitochondrial membranes and cause release of DAMPs andantigenic material. This can have a powerful positive effect on theimmune system's response to cancer cells.

In certain cancer treatments, the immune response is of primaryimportance, e.g. to treat unidentified secondary tumours, to preventformation of metastatic tumours, when surgery or other directintervention is not possible. Different cancers are more or lessimmunogenic and therefore in some scenarios boosting the immune responseto cancer is vital.

Thus, in a further aspect, the present invention provides a peptidiccompound as defined herein for use in the destabilisation of amitochondrial membrane, wherein said use is in the treatment of atumour. This can be regarded as an immunotherapeutic use or treatmentand the tumour will typically be cancerous. Preferred features andembodiments discussed elsewhere in relation to the combination therapiesapply, mutatis mutandis, to this aspect.

In a further aspect the invention provides a composition comprising orconsisting of a peptidic compound of the invention and animmunotherapeutic agent. In a further aspect the invention provides amethod of treating tumours in a patient, said method comprisingadministration of an effective amount of a peptidic compound of theinvention and simultaneous or sequential administration of an effectiveamount of an immunotherapeutic agent.

Alternatively viewed, there is provided a peptidic compound of theinvention and an immunotherapeutic agent for use in the treatment oftumours.

By “immunotherapeutic agent” is meant an agent which modulates theimmune response. Preferably, the immunotherapeutic agent enhances theimmune response against one or more tumor antigens, for example bysuppressing (preferably selectively) Treg cells and or MDSCs and/or byblocking cytotoxic T lymphocyte antigen-4 (CTLA-4), an inhibitoryreceptor expressed on T cells. In all aspects and embodiments of theinvention, the immunotherapeutic agent is preferably cyclophosphamide(CY).

The skilled person would be able to select suitable dosages of theimmunotherapeutic agent.

The immunotherapeutic agent e.g. the chemotherapeutic agent ispreferably administered prior to or simultaneously with the peptidiccompound of the invention, it is most preferred that it is administeredprior to the first administration of the peptidic compound of theinvention. Preferably, it is administered prior to the peptidic compoundof the invention, e.g. at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 daysprior to the peptidic compound of the invention. A single dose ispreferred, but multiple doses may be used, which may be e.g. 1, 2, 3, 4,5, 6, 7, 8, 9, 10 days apart.

“Co-administration” may be simultaneous or sequential and by the same ordifferent routes of administration, e.g. oral and/or parenteral.

There is further provided a product containing a peptidic compound ofthe invention and an immunotherapeutic agent as a combined preparationfor separate, simultaneous or sequential use in treating tumours.

The inventors have also surprisingly found that the treatment of atumour with a peptidic compound of the invention in conjunction with animmunotherapeutic agent can induce an adaptive immunity against furthertumours. The above methods, uses and products (compositions) maytherefore optionally extend to the induction of an adaptive immunityagainst further tumours. Thus, for example, the invention also providesa peptidic compound of the invention and an immunotherapeutic agent foruse in the treatment of tumours in a patient and inducing adaptiveimmunity against tumour growth, development or establishment in saidpatient.

Thus, in a further aspect, there is provided a method of inducingadaptive immunity against tumour growth, development or establishment ina patient, said method comprising administration of an effective amountof a peptidic compound of the invention and simultaneous or sequentialadministration of an effective amount of an immunotherapeutic agent.

Alternatively viewed, the invention provides a peptidic compound of theinvention and an immunotherapeutic agent for use in inducing an adaptiveimmunity against tumour growth, development or establishment.

Alternatively viewed, there is provided the use of a peptidic compoundof the invention and an immunotherapeutic agent in the manufacture of amedicament for use as a vaccine against tumour growth, development orestablishment.

Thus, there is provided a product containing a peptidic compound of theinvention and an immunotherapeutic agent as a combined preparation forseparate, simultaneous or sequential use in inducing an adaptiveimmunity against tumour growth, development or establishment.

The invention also provides a method of vaccinating a subject againsttumour growth, development or establishment through administration of aneffective amount of a peptidic compound of the invention and animmunotherapeutic agent to said patient. Reference to a ‘vaccine’ and‘vaccinating’ both imply a prophylactic effect, thus while there may bebeneficial direct treatment of existing tumours, a significantmotivation in this aspect of the invention is the prevention orreduction in future tumour growth or development.

The invention will now be further described in the following Examplesand with reference to the figures in which:

FIG. 1 is a graph showing the percentage of red blood cell death in aseries of experiments to test peptide LTX-315 at varying concentrations.X-axis shows peptide concentration (μg/ml). Y-axis shows % cell death;

FIG. 2 shows tumour growth in mice re-inoculated with murine A20 B celllymphoma cells compared with growth in the control animals from theinitial study. Diamonds indicate controls from primary studies. Solidsquares indicate re-inoculated mice;

FIG. 3 shows tumour growth in individual mice re-inoculated with murineA20 B cell lymphoma cells having been initially treated with LTX-315.Squares indicate Mouse 1. Triangles (base at bottom) indicate Mouse 2.Triangles (base at top) indicate Mouse 3. Diamonds indicate Mouse 4;

FIG. 4 shows tumour growth in mice re-inoculated with murine CT26WTcolon carcinoma cells compared with growth in the control animals.Diamonds indicate controls from primary studies. Solid squares indicatere-inoculated mice;

FIG. 5 shows tumour growth in individual mice re-inoculated with murineCT26WT colon carcinoma cells having been initially treated with LTX-315.Small squares indicate Mouse 1. Small triangles (base at bottom)indicate Mouse 2. Small triangles (base at top) indicate Mouse 3. Smalldiamonds indicate Mouse 4; Circles indicate Mouse 5. Large squaresindicate Mouse 6. Large triangles (base at bottom) indicate Mouse 7.Large triangles (base at top) indicate Mouse 8. Large diamonds indicateMouse 9;

FIG. 6 shows growth of A20 B-cell lymphomas in irradiated mice thatreceived splenocytes from donor mice showing complete tumour regressionfollowing treatment with LTX-315 (Group 1) or control mice (Group 2)that received splenocytes from naïve donor mice. Squares indicate Group1 (mice that received splenocytes from donors showing completeregression). Diamonds indicate Group 2 (mice that received splenocytesfrom naive donors);

FIG. 7 shows anti-cancer effect of two different treatment regimes onsolid murine A20 tumours (Groups 1 and 2) as compared to non-treatedcontrols (Group 3). Inverted solid triangles (base at top) indicateGroup 1 (treatment). Open squares indicate Group 2 (treatment+adjuvant).Open triangles (base at bottom) indicate Group 3 (control). Order oftumour size (mm²) at Day 21 is (largest to smallest): Group 3, Group 1,Group 2.

FIG. 8 LTX-315 causes rapid cell death in human melanoma cells. 8 a: Invitro cell killing kinetics of LTX-315 (IC₅₀) against human melanomacells after designated time points (5, 15, 30, 45, 60, 90, 120 and 180min) and 8 b: demonstrating the viability of A375 cells after treatmentwith different concentrations of LTX-315 for 60 min. Results from threeexperiments are presented for each time point as mean±SD.

FIG. 9 LTX-315 internalizes and accumulates close to the mitochondria.A375 cells treated for 30 minutes with 1.5 μM fluorescence-labeledLTX-315, and with labeled mitochondria and nucleus. The peptide wasinternalized and detected in close proximity to the mitochondria. A:overlay channels, B: close up, C: mitochondria. D: peptide

FIG. 10 Internalization occurs only in lytic 9-mer compounds such asLTX-315 and not in the non-lytic mock peptide LTX-328. A375 cellstreated with 3 μM LTX-315 or LTX-328 peptide for 60 min. LTX-315 wasdetected in the cytoplasm, while LTX-328 was not internalized. A:LTX-315 60 min incubation, B: LTX-328 60 min incubation.

FIG. 11 LTX-315 treatment causes ultrastructural changes. TEM images ofA375 cells treated with LTX-315 for 60 minutes compared to controlcells. A&D: untreated control cells, B&E: cells treated with 3.5 μM,C:&F cells treated with 17 μM. Magnification 10 000×A-C, 30 000 D-F,scale bar 5 μm.

FIG. 12 ROS generation in LTX-315 induced cell death. A375 cells weretreated with LTX-315 at different concentrations for 15 minutes. Afterpeptide treatment, carboxy-H2DCFDA was added to the samples andfluorescence was analyzed with a fluorescence plate reader. Theexperiment was conducted in duplicate, with bars representing meanfluorescence+−S.D.

FIG. 13 Human melanoma cells treated with LTX-315 release cytochrome-Cin the supernatant. Cytochrome-C release in the supernatant afterLTX-315 treatment of A375 after designated time points (5, 15, 45 min)were determined by ELISA assay.

FIG. 14 HMGB1 is released in the supernatant after LTX-315 treatment.A375 human melanoma cells were treated with 35 μM LTX-315 (top) orLTX-328 (bottom), and cell lysate (L) and supernatant (S) were analyzedwith Western blot, and the LTX-315-treated cells showed a gradualtranslocation from the cell lysate to the cell supernatant. Controlcells were treated with media alone, and showed no translocation after60 minutes.

FIG. 15 Extracellular ATP levels following LTX-315 treatment: A375 cellswere treated with LTX-315 for different time point (5, 15, 30, 45, 60min) at different concentrations or maintained under controlledconditions, and the supernatant was analyzed for the quantification ofATP secretion by luciferase bioluminescence. Quantitative data(mean+−S.D.) for one representative experiment are reported.

FIG. 16 LTX-315 disintegrates the mitochondria membrane. TEM images ofFIG. 16a : human A547 melanoma cells treated with LTX-315 (10 μg/ml) for60 minutes compared to FIG. 16b : control cells.

FIG. 17 Effect of low dose cyclophosphamide alone (17 b), LTX-315 alone(17 c), and the combination of the two therapies (17 d) on tumor growthof murine A20 lymphomas. Palpable lymphoma tumors syngeneic with Balb/cmice were injected i.p. with metronomic cyclophosphamide (day 4) orvehicle (vehicle controls, (17 a)), and with 1.0 mg LTX-315 (20 mg/ml)once per day on day 8, 9 and 10 after tumor challenge.

FIG. 18 Survival curves of metronomic cyclophosphamide alone, LTX-315alone, and the combination of the two therapies on tumor growth againstcontrol, analyzed using a log-rank (Mantel-Cox) test. Results were shownto be significantly different (p<0.0001).

In summary, the Examples below show:

-   Example 1—that LTX-315 is the most potent of the 5 tested compounds    in an in vitro cytotoxic activity study against a panel of 37 human    cancer cell lines.-   Example 2—that LTX-315 is the most potent of the 5 tested compounds    in an in vitro cytotoxic activity study against a panel of 10    lymphoma cell lines.-   Example 3—that LTX 315 has a mean EC₅₀ value greater than 1200 μg/ml    (833 μM) against human red blood cells.-   Example 4—that the anti-tumour activity of LTX-315 resulted in a    complete tumour response in 3 of 7 treated mice for the Group    receiving the optimal dose-   (Group 1) in an investigation into the effect of LTX-315 at    different dose levels on a murine A20 B-cell lymphoma in mice.-   Example 5—that four different LTX-315 treatment regimes demonstrated    a strong anti tumour effect against murine CT26WT (multidrug    resistant) tumours.-   Example 6—that LTX-315 has a broad spectrum of activity against    various multidrug resistant cancer cell lines and, significantly, a    much weaker cytotoxic effect on normal human cells.-   Example 7—that complete tumour regression following initial    treatment of solid murine tumours with LTX-315 resulted in a form of    endogenous long-term protection against growth of the same tumours    following re-inoculation.-   Example 8—that treatment with LTX-315 may confer long term    protection against specific tumours by eliciting an immune response.-   Example 9—that an anti A20 cell immune response have been induced by    the injection of the cocktail of LTX-315 and lysed A20 cells.-   Example 10—that treatment with LTX-315 induces hallmarks of    immunogenic cell death by mitochondria distortion in human melanoma    cells.-   Example 11—that treatment with LTX-315 in combination with CY caused    a complete and long-lasting tumor regression in a high proportion of    test subjects.

EXAMPLE 1

In Vitro Cytotoxic Activity Study of 5 Test Compounds Against a Panel of37 Human Cancer Cell Lines

1. Study Aim

-   -   To determine the concentrations of five novel compounds to        obtain a 50% inhibition of proliferation (IC₅₀) against a panel        of 37 human cancer cell lines.        2. Materials and Methods        2.1. Test Substances        2.1.1. Test Substances    -   Test substances, LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329        (see Table 1) provided in powder form.        2.1.2. Positive Control    -   Triton X-100 was used as positive control, supplied by        Oncodesign (Dijon, France) from Sigma (Saint Quentin Fallavier,        France).        2.1.3. Drug Vehicle and Storage Conditions    -   Compounds were stored at 4° C. Powder was first dissolved in        serum free culture medium (RPMI 1640, Lonza, Verviers, Belgium)        and further diluted using serum-free culture medium to reach        appropriate dilutions. Stock solution was not stored and was        prepared fresh the day of experiment.    -   1% (final concentration) Triton X-100 was obtained by dilution        using culture medium.        2.2. Tumor Cell Lines and Culture Conditions        2.2.1. Tumor Cell Lines

Cancer cell lines and culture media were purchased and provided byOncodesign. The details of the cell lines is presented in Table 1 below.

TABLE 1 Cell lines Origin Source BLOOD CCRF-CEM acute lymphoblasticleukemia, T cells Pharmacell ^(a) CCRF- acute lymphoblastic leukemia, Tcells Pharmacell CEM/VLB HL-60 acute promyelocytic leukemia, AML, ATCC^(b) pluripotent differentiation HL-60/ADR acute promyelocytic leukemia,AML Pharmacell K-562 chronic myeloid leukemia, pleural ATCC effusionmetastasis K-562/Gleevec chronic myeloid leukemia, pleural Oncodesigneffusion metastasis RPMI 8226 myeloma, B cells, Igl-type PharmacellBRAIN SH-SY5Y neuroblastoma, bone marrow ATCC metastasis SK-N-ASneuroblastoma, bone marrow ATCC metastasis U-87 MG glioblastoma,astrocytoma ATCC BREAST MCF-7 invasive ductal carcinoma, pleuralPharmacell effusion metastasis MCF7/mdr adenocarcinoma, pleural effusionPharmacell metastasis MDA-MB-231 invasive ductal carcinoma, pleuralPharmacell effusion metastasis MDA-MB- invasive ductal carcinoma,pleural ATCC 435S effusion metastasis T-47D invasive ductal carcinoma,pleural ATCC effusion metastasis COLON COLO 205 colorectaladenocarcinoma, ascites ATCC metastasis HCT 116 colorectal carcinomaATCC HCT-15 colorectal adenocarcinoma ATCC HT-29 colorectaladenocarcinoma ATCC ENDOTHELIUM HUV-EC-C normal ATCC KIDNEY 786-O renalcell adenocarcinoma ATCC A-498 carcinoma ATCC LIVER Hep G2hepatocellular carcinoma ATCC SK-HEP-1 adenocarcinoma, ascitesmetastasis ATCC LUNG A549 carcinoma Pharmacell Calu-6 anaplasticcarcinoma ATCC NCI-H460 carcinoma, pleural effusion ATCC metastasisOVARY IGROV-1 carcinoma Pharmacell IGROV- carcinoma Pharmacell 1/CDDPNIH:OVCAR-3 adenocarcinoma, ascites metastasis Pharmacell SK-OV-3adenocarcinoma, ascites metastasis Pharmacell PANCREAS BxPC-3adenocarcinoma ATCC PANC-1 carcinoma ATCC PROSTATE DU 145 carcinoma,brain metastasis Pharmacell PC-3 adenocarcinoma, bone metastasis ATCCSKIN A-431 epidermoid carcinoma ATCC Malme-3M Malignant melanoma ATCCSK-MEL-2 malignant melanoma, skin metastasis ATCC ^(a) Pharmacell, Paris^(b) ATCC, Manassas, Virginia, USA2.2.2. Culture Conditions

Tumor cells were grown as adherent monolayers or as suspensions at 37°C. in a humidified atmosphere (5% CO₂, 95% air). The culture medium wasRPMI 1640 containing 2 mM L-glutamine (Lonza, Belgium) and supplementedwith 10% fetal bovine serum (FBS, Lonza). For experimental use, theadherent cells were detached from the culture flask by a 5-minutetreatment with trypsin-versene (Lonza), diluted in Hanks' medium withoutcalcium or magnesium (Lonza) and neutralized by addition of completeculture medium. Cells were counted in a hemocytometer and theirviability was assessed by 0.25% trypan blue exclusion.

Mycoplasma detection was performed using the MycoAlert® MycoplasmaDetection Kit (Lonza) in accordance with the manufacturer'sinstructions. All tested cells were found to be negative for mycoplasmacontamination.

3. Experimental Design and Treatments

3.1. Cell Lines Amplification and Plating

Tumor cells were plated in 96-well flat-bottom microtitration plates(Nunc, Dutscher, Brumath, France) and incubated at 37° C. for 24 hoursbefore treatment in 190 μl of drug-free culture medium supplemented ornot with 10% FBS for adherent or suspension growing cell lines,respectively.

Implantation densities for each cell lines are summarized in Table 2below:

TABLE 2 Implantation Implantation densities densities Cell lines(cells/well) Cell lines (cells/well) CCRF-CEM 25,000 HUV-EC-C 20,000CCRF-CEM/VLB 25,000 786-O 15,000 HL-60 20,000 A-498 15,000 HL-60/ADR20,000 Hep G2 15,000 K-562 20,000 SK-HEP-1 15,000 K-562/IMR 20,000 A54915,000 RPMI 8226 20,000 Calu-6 15,000 SH-SY5Y 20,000 NCI-H460 15,000SK-N-AS 15,000 IGROV-1 15,000 U-87 MG 15,000 IGROV-1/CDDP 15,000 MCF-720,000 NIH:OVCAR-3 15,000 MCF7/mdr 20,000 SK-OV-3 15,000 MDA-MB-23115,000 BxPC-3 15,000 MDA-MB-435S 20,000 PANC-1 15,000 T-47D 15,000 DU145 15,000 COLO 205 15,000 PC-3 15,000 HCT 116 15,000 A-431 15,000HCT-15 15,000 Malme-3M 15,000 HT-29 20,000 SK-MEL-2 15,0003.2. IC₅₀ Determination

The adherent cell lines were washed once with 200 μl FBS-free culturemedium before treatment. Tumor cells were incubated for 4 hours with 10concentrations of compounds in ¼ dilution step with a top dose of 400 μM(range 4×10⁻⁴ to 4×10⁻¹⁰ M), with 1% (final concentration) Triton X-100as positive control and FBS-free culture medium as negative control. Thecells (190 μl) were incubated in a 200 μl final volume of FBS-freeculture medium containing test substances at 37° C. under 5% CO₂.

Three independent experiments were performed, each concentration beingtested in quadruplicate. Control cells were treated with vehicle alone.At the end of treatments, the cytotoxic activity was evaluated by a MTSassay (see § 3.3.).

Dilutions of tested compound as well as distribution to platescontaining cells were performed using a Sciclone ALH 3000 liquidhandling system (Caliper Life Sciences S.A.). According to automate use,a single range of concentrations was tested whatever the cell lines tobe tested. The range was not adapted for each cell line.

3.3. MTS Assay

The in vitro cytotoxic activity of the test substance was revealed by aMTS assay (BALTROP J. A. et al., Bioorg. Med. Chem. Lett. 1991,1:611-614) using a novel tetrazolium compound (MTS,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron couplingreagent named PMS (phenazine methosulfate). Like MTT, MTS is bioreducedby cells into a formazan product that is directly soluble in culturemedium without processing, unlike MTT.

At the end of the cells treatment, 40 μl of a 0.22 μm filtered freshlycombined solution of MTS (20 ml at 2 mg/ml, Ref G1111, Batch 235897, ExpMarch 2009, Promega, Charbonnières, France) and PMS (1 ml at 0.92 mg/ml,Ref P9625, Batch 065K0961, Sigma) in Dulbecco's Phosphate BufferedSaline (DPBS, Ref 17-513Q, Batch 6MB0152, Cambrex), were added in eachwell. Culture plates were incubated for 2 h at 37° C. Absorbency (OD)were measured at 490 nm in each well using VICTOR³™ 1420 multilabeledcounter (Wallac, PerkinElmer, Courtaboeuf, France).

4. Data Presentation

4.1. IC₅₀ Determination

The dose response inhibition of proliferation (IC) was expressed asfollows:

${IC} = {\frac{{OD}_{{drug}\text{-}{exposed}\mspace{14mu}{wells}}}{{OD}_{{drug}\text{-}{free}\mspace{14mu}{wells}}} \times 100}$

The OD values are the mean of 4 experimental measurements.

IC₅₀: drug concentration to obtain a 50% inhibition of cellproliferation.

The dose-response curves were plotted using XLFit 3 (IDBS, UnitedKingdom). The IC₅₀ determination values were calculated using the XLFit3 software from semi-log curves. Individual IC₅₀ determination values aswell as mean and SD values were generated.

4.2. Resistance Index (RI)

Resistance index was calculated using the following formula:

${RI}_{{compound}\mspace{14mu} A} = \frac{{IC}_{50\;{compound}\mspace{14mu} A}\left( {{Resistant}\mspace{14mu}{cell}\mspace{14mu}{line}} \right)}{{IC}_{50\;{compound}\mspace{14mu} A}\left( {{Sensitive}\mspace{14mu}{cell}\mspace{14mu}{line}} \right)}$

Resistance index was calculated for each compound for each couple ofsensitive and resistant cell lines. Individual resistance index wascalculated when IC₅₀ values of both sensitive and correspondingresistant cell lines were determined within same experiment. Inaddition, resistance index was also calculated ratio of mean IC₅₀ valuesobtained during three independent experiments.

5. Results

5.1. LTX-302

All thirty seven human tumor cell lines tested were sensitive to LTX-302compound with IC₅₀ values ranging from 4.83±0.96 μM to 20.09±4.07 μM forT-47D and Hep G2 cell lines, respectively.

Mean IC₅₀ value for LTX-302 compound obtained on the 37 tumor cell lineswas 12.05±4.27 μM with a median value of 11.70 μM. Mean IC₅₀ valueobtained for the normal cell line (HUV-EC-C) was higher than for any ofthe tumor cell lines.

Hematological and lung cancer cell lines were the most sensitive toLTX-302 compound (median IC₅₀ values 7.96 μM (n=7) and 9.02 μM (n=3) forhematological and lung cancer cell lines, respectively) whereas livercancer cell lines were the most resistant (median IC₅₀ value 17.84 μM,n=2).

Activity of LTX-302 compound seemed to be slightly decreased by acquiredresistance towards doxorubicin as exhibited by the RI values of bothHL-60/ADR and MCF-7/mdr cell lines (1.31 and 1.23 for HL-60/ADR andMCF-7/mdr cell lines, respectively). On the contrary, activity ofLTX-302 compound seemed to be increased by acquired resistance towardscisplatin as exhibited by a RI value of 0.33 for IGROV-1/CDDP cell line.

5.2. LTX-313

All thirty seven (37) human tumor cell lines tested were sensitive toLTX-313 compound with IC₅₀ values ranging from 4.01±0.39 μM to18.49±4.86 μM for RPMI 8226 and U-87 MG cell lines, respectively.

Mean IC₅₀ value for LTX-313 compound obtained on the 37 tumor cell lineswas 9.60±3.73 μM with a median value of 8.83 μM. Mean IC₅₀ valueobtained for the normal cell line (HUV-EC-C) was higher than for any ofthe tumor cell lines.

Hematological cancer cell lines were the most sensitive to LTX-313compound (median IC₅₀ value 7.04 μM, n=7) whereas liver cancer celllines were the most resistant (median IC₅₀ value 13.71 μM, n=2).

Activity of LTX-313 compound seemed not to be modified by acquiredresistance towards doxorubicin as exhibited by the RI values ofCCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines (0.76, 1.16 and 1.24for CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines, respectively). Onthe contrary, activity of LTX-313 compound seemed to be increased byacquired resistance towards cisplatin as exhibited by a RI value of 0.49for IGROV-1/CDDP cell line.

5.3. LTX-315

All thirty seven human tumor cell lines tested were sensitive to LTX-315compound with IC₅₀ values ranging from 1.18±0.25 μM to 7.16±0.99 μM forT-47D and SK-OV-3 cell lines, respectively.

Mean IC₅₀ value for LTX-315 compound obtained on the 37 tumor cell lineswas 3.63±1.45 μM with a median value of 3.27 μM. Mean IC₅₀ valueobtained for the normal cell line (HUV-EC-C) was higher than for any ofthe tumor cell lines.

Breast, hematological and lung cancer cell lines were the most sensitiveto LTX-315 compound (median IC₅₀ values 2.45 μM (n=5), 2.60 μM (n=7) and2.83 μM (n=3) for breast, hematological and lung cancer cell linesrespectively) whereas liver cancer cell lines were the most resistant(median IC₅₀ value 5.86 μM, n=2).

Activity of LTX-315 compound seemed to be slightly decreased by acquiredresistance towards doxorubicin as exhibited by the RI values ofHL-60/ADR and MCF-7/mdr cell lines (1.45 and 1.12 for HL-60/ADR andMCF-7/mdr cell lines, respectively). On the contrary, activity ofLTX-315 compound seemed to be increased by acquired resistance towardscisplatin as exhibited by a RI value of 0.50 for IGROV-1/CDDP cell line.

5.4. LTX-320

All thirty seven human tumor cell lines tested were sensitive to LTX-320compound with IC₅₀ values ranging from 3.46±0.22 μM to 16.64±3.15 μM forT-47D and Hep G2 cell lines, respectively.

Mean IC₅₀ value for LTX-320 compound obtained on the 37 tumor cell lineswas 7.58±2.79 μM with a median value of 6.92 μM. Mean IC₅₀ valueobtained for the normal cell line (HUV-EC-C) was higher than for any ofthe tumor cell lines.

Hematological, breast, kidney and brain cancer cell lines were the mostsensitive to LTX-320 compound (median IC₅₀ values 6.04 μM (n=7), 6.60 μM(n=5), 6.60 μM (n=2) and 6.92 μM (n=3) for hematological, breast, kidneyand brain cancer cell lines respectively) whereas liver cancer celllines were the most resistant (median IC₅₀ value 11.46 μM, n=2).

Activity of LTX-320 compound seemed not to be modified by acquiredresistance towards doxorubicin as exhibited by the RI values ofHL-60/ADR and MCF-7/mdr cell lines (0.90 and 1.19 for HL-60/ADR andMCF-7/mdr cell lines, respectively). On the contrary, activity ofLTX-320 compound seemed to be increased by acquired resistance towardscisplatin as exhibited by a RI value of 0.49 for IGROV-1/CDDP cell line.

5.5. LTX-329

All thirty seven human tumor cell lines tested were sensitive to LTX-329compound with IC₅₀ values ranging from 2.43±0.34 μM to 16.90±1.18 μM forT-47D and U-87 MG cell lines, respectively.

Mean IC₅₀ value for LTX-329 compound obtained on the 37 tumor cell lineswas 8.17±3.20 μM with a median value of 7.89 μM. Mean IC₅₀ valueobtained for the normal cell line (HUV-EC-C) was higher than for any ofthe tumor cell lines.

Breast and hematological cancer cell lines were the most sensitive toLTX-329 compound (median IC₅₀ values 4.92 μM (n=5) and 5.26 μM (n=7) forbreast and hematological cancer cell lines respectively) whereas ovariancancer cell lines were the most resistant (median IC₅₀ value 13.37 μM,n=4).

Activity of LTX-329 compound seemed not to be modified by acquiredresistance towards doxorubicin as exhibited by the RI values ofCCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines (0.76, 0.80 and 1.07for CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines, respectively). Onthe contrary, activity of LTX-329 compound seemed to be increased byacquired resistance towards cisplatin as exhibited by a RI value of 0.46for IGROV-1/CDDP cell line.

5.6. General Comments

T-47D breast cancer cell line is the most sensitive cell line whateverthe LTX compound tested.

Hematological cancer cell lines are the most sensitive histological typefor all five compounds tested, liver and ovarian cancer cell lines beingwithin the most resistant cell lines.

All five compounds tested exhibited highest activity on IGROV-1/CDDPcell line (resistant to cisplatin) than on parental IGROV-1 ovariancancer cell line. Doxorubicin resistance seemed to slightly decreaseactivity of LTX compounds.

LTX-315 compound is the most potent compound from the five compoundstested.

6. Conclusions

-   -   All five compounds tested (i.e. LTX-302, LTX-313, LTX-315,        LTX-320 and LTX-329) exhibited cytolytic activity against 37        human cancer cell lines tested with IC₅₀ values in micromolar to        ten micromolar range.    -   LTX-315 compound is the most potent compound tested with IC₅₀        values between 1 and 5 micromolar on all 37 human cancer cell        lines tested.

EXAMPLE 2

In Vitro Cytotoxic Activity Study of 5 Test Compounds Against a Panel of10 Lymphoma Cell Lines

1. Study Aim

-   -   To determine the concentrations of five novel compounds to        obtain a 50% inhibition of proliferation (IC₅₀) against a panel        of 10 lymphoma cell lines.        2. Materials and Methods        2.1. Test Substances        2.1.1. Test Substances    -   Test substances, LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329        (see Table 1) provided in powder form.        2.1.2. Positive Control    -   Triton X-100 was used as positive control and supplied by        Oncodesign (Dijon, France) from Sigma (Saint Quentin Fallavier,        France).        2.1.3. Drug Vehicle and Storage Condition    -   Compounds were stored at 4° C. Powder was first dissolved in        serum free culture medium (RPMI 1640, Lonza, Verviers, Belgium)        and further diluted using serum-free culture medium to reach        appropriate dilutions. Stock solution was not stored and was        prepared fresh the day of experiment.    -   1% (final concentration) Triton X-100 was obtained by dilution        using culture medium.        2.2. Tumor Cell Lines and Culture Conditions        2.2.1. Tumor Cell Lines

Cancer cell lines and culture media were purchased and provided byOncodesign. The details of the cells lines are presented in Table 3below.

TABLE 3 No Cell lines Origin Source BLOOD 1 Daudi Burkitt's lymphoma, Bcells, ATCC ^(a) peripheral blood 2 Hs 445 Hodgkin's lymphoma, lymphnode ATCC 3 KARPAS- Anaplastic large cell lymphoma, DSMZ ^(b) 299 Tcells, peripheral blood 4 Mino Mantle cell lymphoma, peripheral ATCCblood 5 NAMALWA Burkitt's lymphoma, B cells, ATCC peripheral blood 6Raji Burkitt's lymphoma, B cells, DSMZ peripheral blood 7 RamosBurkitt's lymphoma, B cells, ATCC peripheral blood 8 SU-DHL-1 Anaplasticlarge cell lymphoma, DSMZ pleural effusion 9 Toledo Non-Hodgkin's B celllymphoma, ATCC peripheral blood 10 U-937 Lymphoma, histiocytic,macrophage ATCC differentiation, pleural effusion ^(a) American TypeCulture Collection, Manassas, Virginia, USA ^(b) Deutsche Sammlung vonMikroorganismen und Zellkuturen Gmbh, Braunschweig, Germany2.2.2. Culture Conditions

Tumor cells were grown as suspensions at 37° C. in a humidifiedatmosphere (5% CO₂, 95% air). The culture medium for each cell line isdescribed in Table 4 below. For experimental use, cells were counted ina hemocytometer and their viability was assessed by 0.25% trypan blueexclusion.

TABLE 4 Additives Glu- Gluta- Culture FBS cose mine NaPyr Hepes Celllines medium (%) (g/l) (mM) (mM) (mM) Daudi RPMI 1640 10 — 2 1 10 Hs 445RPMI 1640 20 4.5 2 1 10 KARPAS- RPMI 1640 20 — 2 — — 299 Mino RPMI 164015 4.5 2 1 10 NAMALWA RPMI 1640 10 2.5 2 1 10 Raji RPMI 1640 10 — 2 1 10Ramos RPMI 1640 10 — 2 1 10 SU-DHL-1 RPMI 1640 10 — 2 — — Toledo RPMI1640 15 4.5 2 1 10 U-937 RPMI 1640 10 — 2 — —

Mycoplasma detection was performed using the MycoAlert® MycoplasmaDetection Kit (Lonza) in accordance with the manufacturer'sinstructions. All tested cells were found to be negative for mycoplasmacontamination.

3. Experimental Design and Treatments

3.1. Cell Lines Amplification and Plating

Tumor cells were plated in 96-well flat-bottom microtitration plates(Nunc, Dutscher, Brumath, France) and incubated at 37° C. for 24 hoursbefore treatment in 190 μl of drug-free and FBS-free culture medium.

Implantation densities for each cell lines are summarized in Table 5below:

TABLE 5 Implantation densities No Cell lines (cells/well) 1 Daudi 25,0002 Hs 445 25,000 3 KARPAS-299 25,000 4 Mino 25,000 5 NAMALWA 15,000 6Raji 20,000 7 Ramos 20,000 8 SU-DHL-1 25,000 9 Toledo 25,000 10 U-93715,0003.2. IC₅₀ Determination

Tumor cells were incubated for 4 hours with 10 concentrations ofcompounds in ¼ dilution step with a top dose of 400 μM (range 4×10⁻⁴ to4×10⁻¹° M), with 1% (final concentration) Triton X-100 as positivecontrol and FBS-free culture medium as negative control. The cells (190μl) were incubated in a 200 μl final volume of FBS-free culture mediumcontaining test substances at 37° C. under 5% CO₂.

Three independent experiments were performed, each concentration beingissued from quadruplicate. Control cells were treated with vehiclealone. At the end of treatments, the cytotoxic activity was evaluated bya MTS assay (see § 3.3 below).

Dilutions of tested compound as well as distribution to platescontaining cells were performed using a Sciclone ALH 3000 liquidhandling system (Caliper Life Sciences S.A.). According to automate use,a single range of concentrations was tested whatever the cell lines tobe tested. The range was not adapted for each cell line.

3.3. MTS Assay

The in vitro cytotoxic activity of the test substance was revealed by aMTS assay (Baltorp et al.) using a novel tetrazolium compound (MTS,3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron couplingreagent named PMS (phenazine methosulfate). Like MTT, MTS is bioreducedby cells into a formazan product that is directly soluble in culturemedium without processing, unlike MTT.

At the end of the cells treatment, 40 μl of a 0.22 μm filtered freshlycombined solution of MTS (20 ml at 2 mg/ml, Ref G1111, Batch 235897, ExpMarch 2009, Promega, Charbonnières, France) and PMS (1 ml at 0.92 mg/ml,Ref P9625, Batch 065K0961, Sigma) in Dulbecco's Phosphate BufferedSaline (DPBS, Ref 17-513Q, Batch 6MB0152, Cambrex), were added in eachwell. Culture plates were incubated for 2 h at 37° C. Absorbency (OD)were measured at 490 nm in each well using VICTOR³™ 1420 multilabeledcounter (Wallac, PerkinElmer, Courtaboeuf, France).

4. Data Presentation

4.1. IC₅₀ Data was Determined as in Example 1

5. Results

5.1. LTX-302

All ten human lymphoma cell lines tested were sensitive to LTX-302compound with IC₅₀ values ranging from 5.30±2.02 μM to 12.54±3.52 μM forU-937 and Raji cell lines, respectively.

Mean IC₅₀ value for LTX-302 compound obtained on 10 sensitive cell lineswas 8.11±2.44 μM with a median value of 7.53 μM.

5.2. LTX-313

All ten human lymphoma cell lines tested were sensitive to LTX-313compound with IC₅₀ values ranging from 3.21±2.81 μM to 16.08±4.86 μM forRamos and Raji cell lines, respectively.

Mean IC₅₀ value for LTX-313 compound obtained on 10 sensitive cell lineswas 7.05±3.91 μM with a median value of 5.89 μM.

5.3. LTX-315

All ten human lymphoma cell lines tested were sensitive to LTX-315compound with IC₅₀ values ranging from 1.15±0.42 μM to 4.93±1.03 μM forU-937 and Raji cell lines, respectively.

Mean IC₅₀ value for LTX-315 compound obtained on 10 sensitive cell lineswas 3.01±1.36 μM with a median value of 2.93 μM.

5.4. LTX-320

All ten human lymphoma cell lines tested were sensitive to LTX-320compound with IC₅₀ values ranging from 2.22±NA μM to 11.26±3.42 μM forHs 445 and Raji cell lines, respectively.

Mean IC₅₀ value for LTX-320 compound obtained on 10 sensitive cell lineswas 5.03±2.82 μM with a median value of 4.84 μM.

5.5. LTX-329

All ten human lymphoma cell lines tested were sensitive to LTX-329compound with IC₅₀ values ranging from 2.46±NA μM to 8.70±1.70 μM for Hs445 and Raji cell lines, respectively.

Mean IC₅₀ value for LTX-329 compound obtained on 10 sensitive cell lineswas 5.76±2.27 μM with a median value of 5.72 μM.

5.6. General Comments

KARPAS-299 and Raji cell lines are the most resistant cell lineswhatever the LTX compound tested.

Hs 445, Ramos and U-937 cell lines are the most sensitive cell lineswhatever the LTX compound tested.

LTX-315 compound is the most potent compound from the five compoundstested.

6. Conclusions

-   -   All five compounds tested (i.e. LTX-302, LTX-313, LTX-315,        LTX-320 and LTX-329) exhibited cytolytic activity against the 10        human lymphoma cell lines tested with IC₅₀ values in micromolar        range.    -   LTX-315 compound is the most potent compound tested with IC₅₀        values between 1 and 5 micromolar on all 10 human lymphoma cell        lines tested.

EXAMPLE 3

Haemolytic Activity In Vitro

Principle of Test

The haemolytic activity of the peptide LTX-315 against human red bloodcells was measured.

Materials and Methods

Freshly collected human blood was centrifuged at 1500 rpm for 10 minutesin order to isolate the red blood cells. The red blood cells (RBC) werewashed three times with PBS [35 mM phosphate buffer with 150 mM NaCl, pH7.4] by centrifugation at 1500 rpm for 10 minutes, and adjusted to 10%haematocrit with PBS. LTX-315 solutions were added to give a finalconcentration range of the peptide from 1200 μg/ml to 1 μg/ml and an RBCconcentration of 1%. The resulting suspension was incubated withagitation for one hour at 37° C. After incubation the suspension wascentrifuged at 4000 rpm for 5 minutes, and the released haemoglobin weremonitored by measuring the absorbance of the supernatant at 405 nm. PBSwas used as negative control and assumed to cause no haemolysis. 0.1%Triton was used as positive control and assumed to cause completehaemolysis.

Test substance: LTX-315

Reference substances: PBS (negative control) and Triton X-100 (positivecontrol). Components of reaction mixtures: LTX-315, 10% Triton X-100,PBS and RBC (10% haematocrit). Details regarding these substances ispresented in Table 6 below.

TABLE 6 Concentration PBS (μl) RBC (μl) LTX-315/Triton X-100 (μl) Neg.Control 630 70 — Pos. Control 623 70 7 1200 150 50 300 (2 mg/ml stock)1000 200 50 250 (2 mg/ml stock) 500 325 50 125 (2 mg/ml stock) 100 59570 35 (2 mg/ml stock) 50 612.5 70 17.5 (2 mg/ml stock) 10 560 70 70 (0.1mg/ml stock) 1 623 70 7 (0.1 mg/ml stock)Method of Evaluation:

Released haemoglobin was monitored by measuring the absorbance of thesupernatant at 405 nm, and percent haemolysis was calculated by theequation:% Haemolysis=[(A ₄₀₅ LTX-315−A ₄₀₅ PBS)/(A ₄₀₅ 0.1% Triton X-100−A ₄₀₅PBS)]×100

LTX-315 concentration corresponding to 50% haemolysis (EC₅₀) wasdetermined from a dose-response curve.

Results

Mean value of five different experiments with standard deviation arepresented in Table 7 below.

TABLE 7 LTX-315 Concentration Mean cell Standard Number of (μg/ml) death(%) Deviation parallels 1200 37.7 8.1445 3 1000 38.2 9.5760 5 500 20.47.8613 5 100 3.6 1.1402 5 50 1.6 0.5477 5 10 0.6 0.8944 5 1 0.0 0.000 5The data are also represented in FIG. 1. FIG. 1 shows that LTX-315 has amean value of EC₅₀ higher than 1200 μg/ml (833 μM).

EXAMPLE 4

Pharmacodynamic Effects Relative to Murine A20 B-Cell Lymphoma Tumoursin Mice

Principle of Test

The aim of the study was to investigate the effect of LTX-315 atdifferent dose levels on a murine A20 B-cell lymphoma in mice.

Materials and Methods

The administration took place by intratumoural injection of LTX-315dissolved in sterile saline.

Female mice were inoculated subcutaneously in the abdomen with 5 millionmurine A20 cells (ATCC, LGC Promochem AB, Middlesex, England) in avolume of 50 μl. The mice were divided into four groups (see Table 8below for details). The intratumoural treatment was initiated when thetumours had reached the desired size of approximately 5 mm in diameter(minimum of 20 mm²).

Three dose levels of LTX-315, 1 mg (Group 1), 0.5 mg (Group 2) and 0.25mg (Group 3) per injection, were investigated. The volume was 50 μl forall injections. LTX-315 was dissolved in sterile 0.9% NaCl watersolution. This vehicle was used as control (Group 4). All four groupsreceived three injections.

The mice were monitored during the study by measuring the tumours andweighing the animals regularly. The mice were followed until the maximumtumour burden of 125 mm² was reached, or until serious adverse eventsoccurred (i.e. wound formation upon repeated treatments during thefollow up period), then the mice were sacrificed. A caliper was used fortumour size measurements and weighing and physical examination were usedas health control.

Animals: Specific pathogen-free female Balb/c mice, 6-8 weeks old,supplied form Harlan (England, UK)

Conditioning of animals: Animals were kept on standard laboratory chowand water.

Mean body weight, dose, route and treatment schedule is given in Table 8below.

TABLE 8 Number of Initial body weight Schedule Group animals (g; mean ±SE) Treatment Dose Route (Day*) 1 7 20.36 ± 0.56 Once 1 mg in Intra 1,2, 3 daily 50 μl tumour (20 mg/ml) 2 7 19.96 ± 0.38 Once 0.5 mg in Intra1, 2, 3 daily 50 μl tumour (10 mg/ml) 3 9 20.11 ± 0.33 Once 0.25 mgIntra 1, 2, 3 daily in 50 μl tumour (5 mg/ml) 4 7 19.73 ± 0.40 Once 50μl 0.9% Intra 1, 2, 3 daily NaCl in tumour H₂0 *Day 1 is first day oftreatmentResults:

The anti-tumour effect of the various treatments is presented as meantumour size in Table 9 below.

TABLE 9 Mean tumour Mean tumour Mean tumour Mean tumour size (mm²) size(mm²) size (mm²) size (mm²) Treatment at day 1* on day 4 on day 9 on day14 Group 1 25.82 ± 0.80 0  3.70 ± 2.40 12.43 ± 7.87  Group 2 22.03 ±0.63 0 11.41 ± 4.69 61.08 ± 23.84 Group 3 21.25 ± 0.64 20.60 ± 5.71 68.49 ± 12.74 69.42 ± 17.70 Group 4 22.79 ± 0.68 45.51 ± 5.27 57.79 ±4.39 84.70 ± 7.35  *Tumour size prior to start of treatment at first dayof treatment

The degree of tumour response in the different treatment groups issummarised in Table 10 below.

TABLE 10 Free of Tumour Response Tumour at Animal no partial completeRelapse of end of Group response response response Tumour Follow-Up 1 042.8% (3/7) 57.2% (4/7) 25%  42.8% (3/7) 2 0 71.42% 28.57% (2/7)  0%(0/2) 28.57% (2/7) 3 77.77% 22.22%   0% (0/9) NA 0 4  100% NA NA NA NADiscussion/Conclusions

In Group 3, receiving the lowest LTX-315 dose (0.25 mg/dose), a smallinhibitory effect is observed during the first days. In Group 1 andGroup 2, receiving LTX-315 doses of 1.0 mg/dose and 0.5 mg/doserespectively, all animals showed partial or complete tumour response. Itwas found that the anti-tumour activity resulted in a complete tumourresponse in 3 of 7 treated mice for the Group receiving the optimal dose(Group 1).

Generally stronger necrosis and more wound formation were observed inGroup 1 compared to the other two groups. Except from the woundformation no other adverse events or toxic effects were observed ineither of the groups of animals.

Both 1 mg and 0.5 mg of LTX-315 demonstrated a strong and rapid antitumour effect in the first period of the study. However, as the studyprogresses more animals in Group 2 relapses than in Group 1.

EXAMPLE 5

The Effect of LTX-315 on Murine CT26WT Colon Carcinoma Tumours in Mice

Materials and Methods

The administration takes place by intra-tumoural injection of LTX-315dissolved in sterile saline (0.9% NaCl in sterile water).

Each of a total of 40 female mice was inoculated with five millionmurine CT26WT cells (ATCC, LGC Promochem AB, Boras, Sweden)subcutaneously on the abdomen surface in a volume of 50 μl. The micewere divided into five groups, 8 mice in each group. When the tumoursreached the desired size of 20 mm² the treatment by intra tumouralinjection was initiated. Group one was treated solely on day 1, Grouptwo on day 1 and 2, Group three on day 1 and 3 and Group four on day 1,2 and 3. All daily treatments were one single injection of 1.0 mgLTX-315 dissolved in 50 μl (20 mg/ml). Group five was treated with the50 μl of vehicle for LTX-315 (Group 5).

The mice were monitored during the study by measuring the tumours(digital caliper) and weighing the animals regularly. The mice werefollowed until the maximum tumour burden of 125 mm² was reached, oruntil serious adverse events occurred (i.e. wound formation due torepeated injections), then the mice were sacrificed. Weighing andphysical examination were used as health controls.

Animals: Specific pathogen-free female Balb/c mice, 6-8 weeks old,supplied form Harlan (England, UK)

Conditioning of animals: Standard animal facility conditions. Mean bodyweight, dose, route and treatment schedule is given in Table 11 below.

TABLE 11 Number of Initial body weight Schedule Group animals (g; mean ±SE) Treatment Dose Route (Day*) 1 8 19.00 ± 1.087  Once 1 mg in Intra 1daily 50 μl tumour (20 mg/ml) 2 8 19.56 ± 1.087  Once 1 mg in Intra 1, 2daily 50 μl tumour (20 mg/ml) 3 8 19.41 ± 0.8999 Once 1 mg in Intra 1, 3daily 50 μl tumour (20 mg/ml) 4 8 19.00 ± 0.9396 Once 1 mg in Intra 1,2, 3 daily 50 μl tumour (20 mg/ml) 5 8 18.71 ± 0.7868 Once 50 μl 0.9%Intra 1, 2, 3 (control) daily NaCl in tumour H₂0 *Day 1 is first day oftreatmentResults

The anti-tumour effect of the various treatments is presented as meantumour size in Table 12 below.

TABLE 12 Mean tumour Mean tumour Mean tumour Mean tumour size (mm²) size(mm²) size (mm²) size (mm²) Treatment at day 1* on day 6 on day 10 onday 17 Group 1  22.69 ± 0.4070 4.343 ± 2.295 7.171 ± 4.035 3.712 ± 3.712Group 2 22.90 ± 1.155 1.458 ± 1.458 5.058 ± 4.014 6.644 ± 3.430 Group 321.43 ± 1.141 2.983 ± 2.983 10.85 ± 7.553 0.00 ± 0.00 Group 4 24.09 ±1.653 0.00 ± 0.00 0.00 ± 0.00 1.308 ± 1.308 Group 5 21.39 ± 1.683 33.77± 3.168 48.37 ± 7.035 40.64 ± 19.77 *Tumour size prior to start oftreatment at first day of treatment

Complete tumour response was observed in the vast majority of allanimals treated with LTX-315. The degree of tumour response in thedifferent treatment groups is summarised in Table 13 below.

TABLE 13 Free of Tumour Response Tumour at Animal no partial completeRelapse of end of Group response response response Tumour Follow-Up 1 027.5% 62.5% 20% (1/5)  50% (4/8) 2 0 12.5% 87.5% 71% (5/7)  25% (2/8) 312.5% 0 87.5% 29% (2/7) 62.5% (5/8) 4 0 0 100% (8/8) 37.5% 62.5% (5/8) 5100% (8/8) NA NA NA NADiscussion/Conclusions

The treatment was started when the tumours had reached the desired sizeof a minimum of 20 mm² and animals were sacrificed when the tumoursreached the maximum tumour burden of 125 mm².

End of study was defined as day 17 when six out of eight control animals(Group 5) were sacrificed.

All LTX-315 treatment regimes resulted in a strong anti CT26WT-tumoureffect.

Totally 27 of the 32 treated animals were observed with a completetumour response and four with a partial response. Only one animal (inGroup 3) did not have a response to the treatment. The results presentedshow that all four treated groups have very similar overall tumourresponse, the data also indicate that the degree of relapse of tumourwas higher in Group 2 than in Group 1, 3 and 4. In addition feweranimals were observed to be free of tumour at end of follow-up in Group2 (FIG. 2).

Necrosis and complete tumour response was observed in all the treatedgroups. In Group 1 four out of eight animals, in Group 2 two out ofeight animals, in Group 3 five out of eight animals, and in Group 4 fiveout of eight animals showed complete tumour response. At this stage thetumour was completely necrotic and a wound crust formed at the locationof the tumour.

Necrosis at the tumour site was seen in all treatment groups. Generally,animals in Group 2, 3 and 4 showed more necrosis, wound and crustformation than the animals in Group 1 that were given only one injectionof LTX-315. Group 4 animals, which were given three injections, showedthe most necrosis, wound and crust formation. The difference in necrosisbetween Group 1 and Group 4 was quite large but the animals given thehighest number of treatments seemed to cope well. No toxic or otheradverse effects besides local necrotic tissue and wound formation wereobserved in either of the treated groups of animals.

All four treatment regimes of LTX-315 tested demonstrated a strong antitumour effect against murine CT26WT tumours.

The amount of necrosis, wound and crust formation was proportional tothe number of LTX-315 treatments given.

EXAMPLE 6

LTX-315 Activity Against Sensitive and Multidrug-Resistant Cancer Cellsand Normal Human Cells

Characteristics of the cell lines tested are presented in Table 14below.

TABLE 14 Drug Cell line susceptibility Origin IC₅₀ μM HL-60 SensitiveAcute promyelocytic leukemia 2.07 HL-60/ADR Resistant Acutepromyelocytic leukemia 3.01 MCF-7 Sensitive Breast carcinoma 1.94MCF-7/mdr Resistant Breast carcinoma 1.96 IGROV-1 Sensitive Ovarycarcinoma 6.37 IGROV- Resistant Ovary carcinoma 3.19 1/CDDP K-562Sensitive Chronic myeloid leukemia 3.27 K5627/Gleevec Resistant Chronicmyeloid leukemia 2.98 HUV-EC-C — Normal endothelial cells 23 RBC — Redblood cells 833

The above data shows the broad spectrum of activity of LTX-315 againstvarious cancer cell lines and, significantly, a much weaker cytotoxiceffect on normal human cells.

EXAMPLE 7

Re-challenge with murine A20 B-cell lymphoma and murine CT26WT coloncarcinoma cells in mice with complete tumour regression.

This study sought to investigate the effects of tumour growth in animalsthat had previously shown complete tumour regression following treatmentwith LTX-315.

Methods: Female Balb-c mice (n=4), previously treated with LTX-315, 1mg) or (n=9); previously treated with LTX-315 0.5 or 1 mg) werere-inoculated (s.c. in the abdominal area) with either murine A20 B celllymphoma cells or CT26WT colon carcinoma cells (5 million) respectively6 weeks following initial treatment with LTX-315. Tumour growth wasmonitored for up to 36 days following re-inoculation.

Significant inhibition (P<0.006) of tumour growth was observed in all 4mice treated previously with LTX-315 (1 mg) in study R315-03 comparedwith control animals (FIG. 2) and while relapse was seen in 1 animal, 3weeks later, complete tumour regression was observed in the other 3 mice(FIG. 3).

In 9 mice previously treated with LTX-315 (0.5 or 1 mg) inhibition(P<0.01) of tumour growth was observed in comparison with controlanimals (FIG. 3). The sudden drop in tumour size in FIG. 20, after Day18, is explained by the death of 6 animals bearing large tumours.Inhibition was observed in 7 mice and complete regression in 2 of theanimals (FIG. 5).

Taken together these data suggest that complete tumour regressionfollowing initial treatment of solid murine tumours (murine A20 B celllymphoma or CT26WT colon carcinoma) with LTX-315 resulted in a form ofendogenous long-term protection against growth of the same tumoursfollowing re-inoculation. Inhibition of tumour growth was morepronounced in animals bearing A20 B cell lymphoma tumours when comparedwith animals bearing CT26WT colon tumours.

EXAMPLE 8

Immunological effects of LTX-315 in a murine A20 B-cell lymphoma model.An in vivo adoptive spleen cell transfer pilot study.

This study was undertaken to investigate whether the long-termprotection against growth of the same tumours following re-inoculationin animals observed in study R315-33 could be passively transferred tonaive recipients via spleen cells taken from LTX-315-treated donoranimals.

Ten female Balb/c mice (n=32) were each inoculated with A20 cells (5million in 50 μL s.c.) on the abdominal surface. Once tumours hadreached 20 mm² they were injected with LTX-315 (1 mg) injectedintratumourally, once daily for 3 days, in a volume of 50 μL. Tumoursize (mm²) and body weight were subsequently monitored and a furtherinjection of LTX-315 was given if any tumour re-growth was observed.Subsequently, mice showing complete tumour regression were sacrificedand used as donors for transfer of splenocytes while naive donor micewere used as controls. Spleens from donor mice were excised and cellsisolated. Naive receiver mice were irradiated and divided into 2 groups.Group 1 received isolated splenocytes from cured mice, whereas group 2received isolated splenocytes from naive mice. Freshly prepared cellswere injected (20×106 per 100 μl) via the tail vein. Twenty four hourslater receiver mice were inoculated with 5 million murine A20 B-celllymphoma cells on the abdominal surface as described above. Tumour sizeand body weight were monitored until the maximum tumour burden of ˜125mm² was reached, or a serious adverse events occurred (i.e. woundformation due to tumour tissue necrosis) at which point mice weresacrificed.

Inhibition of tumour growth was observed in irradiated mice thatreceived splenocytes isolated from animals that had shown completetumour regression following treatment with LTX-315 when compared withcontrol animals that received splenocytes from naive donors (FIG. 6). Itwas also noted that there was a difference in the colour and texture ofthe tumours in recipients of splenocytes from LTX-315-treated micesuggesting an immediate inflammatory response.

Based on these observations, the data provides evidence for an adaptiveimmune response in the animals that received splenocytes from animalsthat previously showed complete regression of A20-B lymphoma tumoursfollowing treatment with LTX-315. This data suggests that treatment withLTX-315 may confer long term protection against specific tumours byeliciting an immune response.

EXAMPLE 9

The objective of the study was to investigate the anti-cancer effect ofprophylactic vaccination with A20 lymphoma cells lysed by 10 mg/mlLTX-315:

-   -   (i) alone; and    -   (ii) in combination with 20 mg/ml LTX-315 injected at the        vaccination site prior to the vaccine.

In total, two different treatment regimens were used.

Administration was by subcutaneous injection of LTX-315 dissolved ingrowth media containing A20 lymphoma cells. The cell-LTX-315 “cocktail”was left for 30 min prior to injection in order to assure complete lysisof the cancer cells.

Group 1 (“vaccine”) mice were injected subcutaneously on the abdomensurface with 50 μl of a “cocktail” of ten million murine A20 cells(ATCC, LGC Promochem AB, Boras, Sweden) and 10 mg/ml LTX-315 (“A20lysate”). Group 2 (“vaccine+adjuvant”) mice were treated as per Group 1,but in addition were given 25 μl of 20 mg/ml LTX-315 subcutaneously atthe site of vaccination 5 minutes prior to the A20 lysate injection.Group 3 (“control”) mice received no treatment.

Six weeks after the treatment, all mice were inoculated with 5 millionviable A20 B-cell lymphoma cells subcutaneously on the abdomen surfacein a volume of 50 μl.

The mice were monitored during the study by measuring the tumour sizeand weighing the animals regularly. The mice were followed until themaximum tumour burden of ˜130 mm² was reached, at which point the micewere sacrificed.

Materials and Methods

Animals: Specific pathogen-free female Balb/c mice, 6-8 weeks old,supplied from Harlan Laboratories (England, UK; www.harlan.com)

Conditioning of animals: Standard animal facility conditions at theUniversity of Tromsø.

Test substance: Murine A20 cells lysed by LTX-315 (Lot 1013687), andLTX-315 (Lot 1013687) alone

Test substance preparation: 10×10⁶ A20 cells were added to a 50 μl 10mg/ml LTX-315/vehicle (“A20 lysate”). The test substance was ready foruse 30 minutes after mixing. LTX-315 alone was dissolved in 0.9% NaCl insterile H₂O

Vehicle: RPMI-1640 w/2 mM L-glutamine or 0.9% NaCl in sterile H₂O

Reference substances: Not applicable

Treatment of controls: Not applicable

Method of evaluation: Tumour size measurements and health control byweighing and examination

Additional data regarding method: A digital caliper was used for tumoursize measurements and weighing and physical examination were used ashealth control

Mean body weight, dose, route and treatment schedule are shown in Table15 (below).

TABLE 15 No of Initial body weight Cell numbers Group animals (g; mean ±SE) Treatment and dose Route 1 8 17.31 ± 0.3815 Once 10 × 10⁶ A20 cellsin Subcutaneous 50 μl LTX-315 (10 mg/ml) 2 8 17.14 ± 0.4633 Once 0.25 μlLTX-315 (20 Subcutaneous mg/ml) + 10 × 10⁶ A20 cells in 50 μl LTX-315(10 mg/ml) 3 7 17.29 ± 0.3020 Not Not applicable Not applicable treatedResults:

The anti cancer effect of the various treatments is presented as meantumour size in Table 16 below and a graphical presentation of the datais provided in FIG. 7. In Table 16, Day 1 was the day of inoculation ofviable A20 cells six weeks post-vaccination.

TABLE 16 Mean tumour Mean tumour Mean tumour Mean tumour size (mm²) size(mm²) size (mm²) size (mm²) Treatment at day 4 on day 11 on day 16 onday 21 Group 1 9.515 ± 1.528 20.44 ± 6.191 36.21 ± 10.30 55.89 ± 15.27Group 2 7.315 ± 2.231 17.13 ± 5.078 29.13 ± 7.903 47.16 ± 13.54 Group 310.25 ± 3.100 34.49 ± 8.298 56.04 ± 8.339 82.89 ± 14.06Discussion/Conclusions:

The inoculation of viable A20 B-cell lymphoma cells was accomplished 6weeks after the treatment was given (day 1) and the animals weresacrificed when the tumours reached the maximum allowed tumour burden of˜130 mm².

The results show that the tumours developed more slowly in bothLTX-315/A20-lysate treatment Groups as compared to the control Group.The median survival of Group 1 was 28 days, 33 days for Group 2, and 25days for the control group (Group 3). Increase in median survival was12% for Group 1 and 35% for Group 2 as compared to the control group(Group 3).

The data indicate a prolonged survival of the treated groups compared tothe untreated control group. On day 34, when the last animal in thecontrol group was sacrificed, 50% of the animals in Group 2 were stillalive while 37.5% of the animals in Group 1 were still alive. End ofstudy was defined as day 60. At this time-point, a total of 3 of the 16treated animals had a complete regression of an initially developingtumour and were tumour free. At the end of the study 25% of animals fromGroup 1, and 12.5% of animals from Group 2 were observed to be tumourfree.

Macroscopically there were morphological differences between the treatedgroups (Group 1 and 2) compared to the non-treated control group (Group3). The developing tumours in the two treatment groups were observed tobe whiter and harder than the tumours observed in the control group.This finding together with the slower growth rate of the tumoursindicates that an anti-A20 cell immune response was induced by thevaccination with the cocktail of LTX-315 and lysed A20 cells.

Hence, LTX-315 may have a dual use by lysing the tumour cells andinducing release of danger signals from normal cells at the injectionsite.

EXAMPLE 10

In this study, we investigated the tumoricidal effect of LTX-315 onhuman melanoma cells. The peptide internalized and was shown inassociation with mitochondria, ultimately leading to a lytic cell death.The LTX-315 peptide was designed to treat solid tumors with intratumoralinjections through a two-stage mode of action: the first is the collapseof the tumor itself, while the second is the released damage-associatedmolecular pattern molecules (DAMPs) from the dying tumor cell, which caninduce a subsequent immune protection against recurrences andmetastastis.

Material and Methods

Reagents

LTX-315 and LTX-328 (K-A-Q-Dip-Q-K-Q-A-W-NH₂) (SEQ ID NO:43) were madeon request by Bachem AG (Bubendorf, Switzerland) and Innovagen (Lund,Sweden), respectively. LTX-315 Pacific Blue and LTX-328 Pacific Bluewere purchased on request from Innovagen (Lund, Sweden) Norud (Tromso,Norway), respectively.

Cell Culture

The A375 cell line A375 (ECACC, 88113005) is a human malignant melanomaderived from patient material, and was purchased from Public HealthEngland (PHE Culture Collections, Porton Down, Salisbury, UK). Cellswere maintained as monolayer cultures in high glucose 4.5% DMEMsupplemented with 10% FBS and 1% L-glutamine, but not as antibiotics(complete media). The cell line was grown in a humidified 5% CO₂atmosphere at 37° C., and was regularly tested for the presence ofmycoplasma with MycoAlert (Lonza).

In Vitro Cytotoxicity, MTT-Assay

The cytotoxic effect of LTX-315 was investigated using the colorimetricMTT viability assay as described in Eliassen et al. (2002), 22(5): pp2703-10. The A375 cells were seeded at a concentration of 1×10⁵ cells/mlin a volume of 0.1 ml in 96-well plates, and allowed to adhere in acomplete growth media overnight. The media was then removed and thecells were washed twice in serum-free, RPMI-1650 media, before addingLTX-315 dissolved in serum-free RPMI at concentrations ranging from2.5-300 μg/ml, and incubated for 5-180 minutes. Cells treated with aserum-free RPMI were used as negative control cells, while cells treatedwith 1% Triton X-100 in serum-free media were used as a positivecontrol. The final results were calculated using the mean of threeexperiments, each with triplicate wells.

Confocal Microscopy

Live Cell Imaging with Unlabeled Cells—

A375 cells were seeded at 10,000 cells/well in a complete media in NuncLab-Tec 8-wells chambered covered glass (Sigma) precoated with 25 μg/mlhuman fibronectin (Sigma) that were allowed to adhere overnight. Cellswere washed twice with a serum-free RPMI, treated with peptide dissolvedin RPMI and investigated using Bright on a Leica TCS SP5 confocalmicroscope, with a 63×/1.2 W objective. The microscope was equipped withan incubation chamber with CO₂ and temperature control.

Fixed Cells, Mitotracker—

Cells were seeded as for live cell imaging, and treated with MitotrackerCMH2XROS (Invitrogen) at 100 nm for 15 minutes prior to peptidetreatment. Cells were treated with 17 μM LTX-315, with negative controlserum-free RPMI only. After 60 min of incubation, cells were analyzedusing a Zeiss microscope. All confocal imaging experiments weresubsequently conducted at least twice with similar results.

Fixed Cells, Fluorescence-Labeled Peptide—

Subconfluential A375 cells were seeded at 8,000 cells/well as above, andtransfected on the second day using the Lipofectamine LTX with Plustransfection reagents (Invitrogen) following the manufacturer'sprotocol. The mitochondria were labeled using the pDsRed2-Mito, and thenucleus was labeled using the GFP-Histon2B plasmid (Imaging Platform,University of Tromsø). A day after transfection, cells were washed twicewith serum-free RPMI, and treated at different concentration andincubation periods with LTX-315 Pacific Blue or LTX-328 Pacific Blue.LTX-315 PB exhibited a similar cytotoxic profile as the unlabeledLTX-315 as determined by MTT assay. Control cells were treated withunlabeled LTX-315 and also with serum-free RPMI only. After incubation,cells were fixed with 4% paraformaldehyde in PBS, and the wells werecovered with Prolong Gold antifade (Invitrogen). Cells were furtheranalyzed by use of a Leica TCS SP5 confocal microscope, with a 693, 1.2W objective. Pacific Blue, GFP and Ds Red were excited using UV, with488 and 561 lasers, and fluorescence channels were sequentially detectedusing the following band passes: UV: 420-480 nm (with attenuation), 488:501-550 nm and 561: 576-676 nm.

TEM Electron Microscopy

A375 cells were seeded at 1×10⁵ cells per well in 6-well plates andallowed to grow for three days to optimize membrane structures in theculture, and the media was changed on the second day. Cells were washedtwice in serum-free RPMI before being treated with LTX-315 dissolved inserum-free RPMI at 5, 10 and 25 μg/ml, with serum-free RPMI as anegative control. Cells were then washed with PBS twice before fixationfor 24 hours in 4° C. with 4% formaldehyde and 1% gluteralaldehyde in aHepes buffer at pH 7.8. Dehydration and post-fixation protocols includedincubation in a 5% buffered tannic acid and incubation in a 1%osmium-reduced ferrocyanide. Ultrathin sections were prepared, anduranyl acetate (5%) and Reynolds's lead citrate were used for stainingand contrasting. Samples were examined on a JEOL JEM-1010 transmissionelectron microscope, and images were taken with an Olympus Moradaside-mounted TEM CCD camera (Olympus soft imaging solutions, GmbH,Germany).

Fluorescence Measurement of Reactive Oxygen Species (ROS)

A DCFDA cellular reactive oxygen species detection assay kit waspurchased from Abcam®, and A375 cells seeded in a 96-well Costar blackclear bottom plate with 20,000 cells per well incubated in 37° C. 16hours prior to DCFDA assay. Cells were washed with a 100 μL/well ofpre-warmed PBS one time, and incubated with 20 μM of DCFDA in a buffersolution supplied with the kit at 37° C. in a cell culture incubator for45 min, and then washed again with a buffer solution of 100 μL/well. Thecells were then stimulated with a 100 μL/well LTX-315 peptide dissolvedin a buffer solution at concentrations of 17 μM for 30 min, and cellsnot treated were used as a negative control. The fluorescence intensitywas determined at an excitation wavelength of 485 nm and an emissionwavelength of 530 nm on a FLUOstar Galaxy plate reader.

Release of High Mobility-Group Box-1 (HMGB1)

A375 cells were seeded with 3×10⁵ cells/well in 6-well plates in acomplete media, and allowed to adhere overnight. Cells were treated withLTX-315 or LTX-328 at 35 μM, and incubated at 37° C. and 5% CO₂ fordifferent time points (5, 10, 15, 30, 60 min), and negative controlswere serum-free RPMI-1650. Supernatants (S) were collected andcentrifuged at 1,400 g for five minutes, and cell lysates (L) wereharvested after washing with PBS twice and then subsequently lysed usinga 4× Sample buffer (Invitrogen, number), 0.1 M DTT (Sigma number) andwater. Supernatants were concentrated using Amicon Ultra 50K centrifugalfilters (Millipore UFC505024), and the cell lysate was sonicated. Bothsupernatants and lysate were boiled and resolved in a 10% sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then electrotransferred to a polyvindiline difluoride (PVDF) membrane (Millipore).The membrane was blocked in 5% milk and incubated with the HMGB1antibody (rabbit, polyclonal, abcam ab 18256); the membrane was thenrinsed several times with TBST, incubated with a horseradish peroxidase(HRP)-conjugated secondary antibody (abcam ab6721), rinsed again withTBST and then developed using WB Luminol Reagent (Santa CruzBiotechnology, Heidelberg, Germany).

Release of Cytochrome-C

A375 cells were seeded as with HMGB1 studies, and treated with 35 μM fordifferent time points (5, 15, 45). Supernatants were collected andconcentrated as with HMGB1 studies, and samples from the supernatantswere analyzed using a 4.5 hour solid form Cytochrome C—Elisa kit (R&DSystems, USA, #DCTCO) following the manufacturer's description. Shortlythereafter, a 50% diluted sample was analyzed and the optical densitywas determined using a microplate reader set at 450 nm, and this readingwas then subtracted from the reading at 540 nm. A standard curve wasgenerated for each set of samples assayed. Samples were run in fourparallels, and the cytochrome-c released into the supernatant wasexpressed as a fold over the level of cytochrome-c in the supernatant ofuntreated cells.

Release of ATP

The supernatant of LTX-315-treated A375 cells was analyzed using anEnliten ATP luciferase assay kit (Promega, USA). Cells were then seededas with an ROS assay, and treated with LTX-315 in different incubationtimes, from 1 to 15 minutes with two parallels, which was then conductedthree times. Negative controls were untreated A375 cells exposed toserum-free media alone. Samples were diluted at 1:50 and 1:100, andanalyzed with a Luminoscan RT luminometer according to themanufacturer's protocol.

Statistical Analysis

All data represent at least two independent experiments with at leasttwo parallels, which were expressed as the mean±SD. Cytochrome-C releaseand ATP release data was compared using one-way ANOVA and a multiplecomparison test, and we considered the P-value <0.05 to indicatestatistical significance.

Results

Cytotoxic Effect of LTX-315 on Melanoma Cells

To investigate the effect of LTX-315 on A735 melanoma cells in vitro, wedetermined the IC-50 values for the peptide by a cell viability assayMTT at different incubation times. The IC-50 value was 30 μM after onlyfive minutes of incubation, and progressed to 14 μM after 90 minutes.Further incubation up to 180 minutes did not offer any additional effect(FIG. 8).

LTX-315 Treatment Causes Rapid Cell Lysis

We next wanted to assess the cell morphology of A735 melanoma cellstreated with LTX-315. Cells were treated with LTX-315 at an IC-50 value,and investigated by bright field confocal microscopy. Treated cellsdisplayed a rapid change from a normal epithelial morphology to a totalcollapse of the cells with an extrusion of cytoplasmic content, whichwas preceded by a rounding up of the cell (data not shown). Thesechanges typically occurred within 15-60 minutes at an IC-50 value in themajority of the cells.

LTX-315 Internalizes and Targets the Mitochondria

To investigate the internalization and fate of the peptide within thecells, LTX-315 was labeled with Pacific Blue and incubated with cells atconcentrations of 3 μM and 1.5 μM, respectively. The labeled LTX-315rapidly penetrated the plasma membrane and at 1.5 μM, the peptide showedan accumulation around the mitochondria after 30 minutes of incubationbut was not detected in the cell nucleus (FIG. 9). The labeled non-lyticmock-sequence peptide LTX-328 did not demonstrate any internalization atany concentration or incubation time tested (FIG. 10).

LTX-315 Induces Ultra-Structural Changes in Cells

We further evaluated the ultrastructural changes in treated cells byperforming transmission electron microscopy (TEM), in which A375 cellswere treated with either peptides dissolved directly in media or inmedia alone. A significant number of the cells treated with a lowconcentration (3.5 μM) of the LTX-315 peptide for 60 minutes showedvacuolization, as well as some altering of the mitochondrial morphology(FIG. 11). The mitochondria appeared to be less electron-dense, alsoexhibiting some degree of reorganization, with the cristae lying furtherapart or not visible at all. The number of necrotic cells in thesesamples was less than 5%. In these low concentrations, vacuolization ofthe cytoplasm was observed. Another common finding in these samples wereperipherally placed vacuoles, which were lined with a single membranelayer containing a homogenous material (FIG. 11B). When cells weretreated with higher concentration (17 μM) for 60 min, approximately 40%of them displayed a necrotic morphology with a loss of plasma membraneintegrity (FIGS. 11C&E). The cells that were still intact displayed agreat heterogeneity, from a normal appearance with microvilli to a roundappearance, with mitochondria clearly affected. In this highconcentration, only 4% of the cells investigated displayedvacuolization, and chromatin condensation was not visible in thismaterial at any peptide concentration tested. These results demonstratethat LTX-315 kills the tumor cells with a lytic mode of action, whilelower concentrations cause the cells to undergo ultrastructure changes,such as vacuolization and an altered mitochondrial morphology. Moreover,no significant morphological changes suggestive of apoptotic cell deathwere observed.

In a separate experiment, exposure of LTX-315 at 10 μg/ml to human A547cells (an ovarian melanoma cell line) led to disintegration of themitochondrial membrane (FIG. 16).

LTX-315 Treatment Leads to Extracellular ATP Release

DAMPs are molecules that are released from intracellular sources duringcellular damage. DAMPs can initiate and perpetuate an immune responsethrough binding to Pattern Recognition Receptors (PRRs) on AntigenPresenting Cells (APCs). Among commonly known DAMPs are ATP, HMGB1,Calreticulin, Cytochrome C, mitochondrial DNA and Reactive oxygenspecies (ROS). We next wanted to investigate whether ATP was releasedinto the supernatant from cells treated with LTX-315. Hence, thesupernatant from treated and non-treated cells analyzed using luciferasedetection assay. As shown in FIG. 15, ATP was detected in thesupernatant as early as after 5 minutes of treatment with LTX-315, andthe release was concentration-dependent.

LTX-315 Treatment Induces Cytochrome-C Release in Supernatant

To assess whether LTX-315-treated cells released cytochrome-C into themedium, A375 cells were treated with LTX-315 at 35 μM at different timepoints (5, 15, 45 min). The supernatant was subsequently analyzed usingan ELISA assay. Cells treated with 35 μM value had three times morecytochrome-C in the supernatant compared to untreated control cells. Theincrease in cytochrome-C was detected after only five minutes oftreatment, and there was also an increase after 15 and 45 minutes ofpeptide treatment, respectively (FIG. 13).

LTX-315 Treatment Leads to Extracellular HMGB1 Release

HMGB1 is a non-histone, chromatin-binding nuclear protein. Oncepassively released from necrotic cells, HMGB1 is able to trigger thefunctional maturation of dendritic cells, cytokine stimulation andchemotaxis among several immunopotentiating effects.

HMGB1 is normally found in the cell nucleus and would be expected in acell lysate of healthy cells, though not in the culture media(supernatant). In order to assess the release of HMGB1 fromLTX-315-treated cells, we measured the translocation and free HMGB1 fromthe nuclear compartment within the cell lysate into the cellsupernatant.

Both cell lysate and the cell supernatant of LTX-315- andLTX-328-treated A375 melanoma cells were analyzed using a Western blot.Cells were treated with 35 μM of either LTX-315 or LTX-328, with agradual translocation from the cell lysate to the supernatant detectedin the LTX-315-treated melanoma cells, but not in the cells treated withthe mock sequence peptide LTX-328 or a serum-free medium only (FIG. 14).

LTX-315 Treatment Causes the Production of Reactive Oxygen Species (ROS)in A375 Melanoma Cells

The ROS generation following LTX-315 treatment was measured by CH2DCFDAfluorometric assay. Significant amounts of ROS were generated after 15minutes of incubation with LTX-315, and the ROS levels wereconcentration-dependent (FIG. 12).

Discussion

LTX-315 labeled with the fluorescent molecule Pacific Blue wasinternalized within minutes after incubation with A375 melanoma cells,and was distributed in the cytoplasm (FIG. 9). At low concentrations,accumulation of the peptide around the mitochondria was evident, whereasat higher concentrations the peptide was more spread within thecytoplasm and accumulated in circular structures closer to the cellmembrane (FIG. 10). If the peptide attacks the mitochondrial membrane, adecrease or even a total collapse of the mitochondrial membranepotential would be expected. A confocal imaging of cells with themembrane potential-dependent mitochondrial stain Mitotracker CMXh2ROSshowed a loss of mitochondrial signal a short time after peptidetreatment (data not shown). The loss of the signal shows that thepeptide interaction with the mitochondria causes a loss of mitochondrialmembrane potential, which is crucial for the mitochondria's mostimportant cellular functions. An altered mitochondrial morphology wasalso demonstrated with TEM. Cells treated with LTX-315 for 60 minuteshad less electron-dense mitochondria with an altered organization of thecristae, as well as vacuolization within the mitochondria compared tountreated cells (FIG. 11). Furthermore, vacuolization was evident inapproximately 20% of cells treated with 3.5 μM of LTX-315. When themitochondria are dysfunctional, free oxygen radicals (ROS) may beformed, and by using fluorometric assays we demonstrated ROS formationwithin a few minutes after peptide treatment (FIG. 12).

In this study, we demonstrate that treatment with the LTX-315 peptidecauses an increase in ROS levels in A375 melanoma cells after treatment.One explanation for these higher levels of ROS following peptidetreatment could be that the peptide enters the cells and targets themitochondria, and the dysfunctional mitochondria then releases ROS.Through an ELISA assay, we detected the release of cytochrome-C in thesupernatant of peptide-treated cells after only a few minutes oftreatment (FIG. 13). Cytochrome-C is a mitochondrial protein releasedfrom the intermembrane space and into the cytosol when the outermitochondrial membrane is perturbed, and by binding to the apoptoticprotease activating factor-1 (Apaf-1) it is also a part of the apoptoticcascade that eventually leads to cell death by apoptosis. However, ifcytochrome-C is found in the extracellular space, it has been reportedto act as a pro-inflammatory mediator, thus activating NF-kB andinducing cytokine and chemokine production. The transition of HMGB1 fromthe cellular compartment to the extracellular compartment was detectedusing a western blot (FIG. 14). When the nuclear protein HMBG1 isreleased into the extracellular fluid, it functions as a DAMP, and canbind to both the PRR TLRs and to the RAGE receptors; the activation ofthese may lead to a number of inflammatory responses such as thetranscription of pro-inflammatory cytokines. We also detected ATPreleased in the supernatant after peptide incubation (FIG. 15), andpresented extracellularly it functions as a DAMP by activating thepurinerg P2RX7 receptors on the DC. This receptor not only functions asa pore that opens for small cationic and later bigger molecules afterbinding to ATP, its activation also causes the processing and release ofthe pro-inflammatory cytokine IL-1β.

In summary, our data suggests that LTX-315 induces lytic cell death incancer cells, not only by direct attack on the plasma membrane, but alsoas a result of an injury to vital intracellular organelles after theinternalization of the peptide at concentrations too low to cause animmediate loss of plasma membrane integrity. We demonstrate that thepeptide treatment causes the release of several DAMPs such as CytC, ATP,HMGB1 and ROS. The DAMPs may affect the cellular integrity of thedamaged cells in several ways, but are also associated with so-calledimmunogenic cell death. The release of tumor-specific antigens into theextracellular compartment, together with potent immune stimulatorymolecules (DAMPs) such as ATP, CytC and HMGB1, can give a strong immuneresponse. In turn, these factors will lead to a maturation andactivation of DCs and other accessory cells of the adaptive immunesystem.

EXAMPLE 11

Introduction

The notion that the successful treatment of cancer is withinpersonalized medicine, and requires a combination of differentmodalities to maximize immune engagement and activation, is widelyaccepted. At higher doses cyclophosphamide (CY) can causeimmunosuppression. However, at low doses (metronomic) the drug can leadto enhanced immune responses against a variety of antigens, morespecifically through the selective suppression of inhibitory cellsubsets including MDSCs and Treg cells. CY can also cause dendritic cellactivation through the release of HMGB1 and ecto-CRT, which has knock-oneffects in terms of pro-inflammatory cytokine production and T cellproliferation. A single i.p. injection of 2 mg CY was shown to lead to areduction in the spleen cells within 24 hours, and with a low-point (50%drop) on the fourth day after CY administration. Relative numbers ofCD4+ and CD8+ T cells increased in spleens and LNs after CYadministration, whereas the relative number of CD19+ and Tregs decreasedsharply.

Principle of Test

Investigate the potential synergistic effects of combining LTX-315 withmetronomic CY in an A20 lymphoma model.

Test substance: LTX-315 (batch number 1037915, correction factor 76.5%,supplied by Lytix Biopharma). Sendoxan (cyclophosphamide monohydrate),purchased at the hospital pharmacy.

Vehicle LTX-315: Saline (0.9% NaCl in sterile H₂O).

Vehicle Sendoxan: Saline (0.9% NaCl in sterile H₂O).

Vehicle control: Saline (0.9% NaCl in sterile H₂O).

Method of Evaluation

Animals:

Female Balb/c wild-type mice, 5-6 weeks old, were obtained from CharlesRiver, United Kingdom. All mice were housed in cages in a pathogen-freeanimal facility according to local and European Ethical Committeeguidelines.

Tumor Treatment:

Tumor cells were harvested, washed in RPMI-1640 and injectedintradermally (i.d.) into the right side of the abdomen in Balb/c mice(5×10⁶ A20 cells per mouse/50 μl RPMI-1640). When animals obtainedpalpable tumors (20-30 mm²) animals were injected i.p. with a singledose of CY (day 4) dissolved in saline (2.0 mg CY/500 μl saline). On day8 peptide treatment was initiated using single i.t. injections ofLTX-315 (1.0 mg LTX-315/50 μl saline) once a day for 3 consecutive days,and the vehicle control was saline only (0.9% NaCl in sterile H₂O).Tumor size was measured using an electronic caliper and expressed as thearea of an ellipse [(maximum dimension/2)×(minimum dimension/2)×π].Animals were then euthanized when the product of the perpendicular tumordimensions reached 130 mm² or when tumor ulceration developed.

Results

A20 lymphomas have been shown to be difficult to treat due to the tumorgrowth pattern (viscous and undefined tumor) and its metastaticpotential. LTX-315 induced complete regression of palpable A20 tumors inone of the animals and a partial response in the remainder of theanimals, following intratumoral injection. To investigate thesynergistic effect of LTX-315 in combination with metronomic CY, Balb/cmice with A20 lymphomas were treated with a single i.p. injection of CY(2 mg/500 μl) on day 4 and i.t. injections with LTX-315 (1 mg/50 μl) forthree consecutive days (FIGS. 17 and 18). Controls were injected i.p.and i.t. with vehicle equivalent to the volume of the correspondingtreatment. It is noteworthy that the tumors in the LTX-315 alone groupwere larger when treatment was initiated compared to the group receivingboth LTX-315 and metronomic CY, as the metronomic CY inhibited the tumorgrowth for 7-10 days.

Conclusion

A majority of the animals treated with LTX-315 in combination withmetronomic CY experienced a complete and long-lasting tumor regressionand were tumor-free 4 weeks post-treatment.

It will be appreciated that it is not intended to limit the presentinvention to the above specific embodiments only, numerous embodiments,modifications and improvements being readily apparent to one of ordinaryskill in the art without departing from the scope of the appendedclaims.

The invention claimed is:
 1. A method of treating a tumour in a subjectin need thereof, comprising the administration to said subject of apharmaceutical composition consisting essentially of an effective amountof a cytotoxic chemotherapeutic agent that enhances an immune response,a compound having the following characteristics: a) consisting of 9amino acids in a linear arrangement; b) of those 9 amino acids, 5 arecationic and 4 have a lipophilic R group; c) at least one of said 9amino acids is a non-genetically coded amino acid; and optionally d) thelipophilic and cationic residues are arranged such that there are nomore than two of either type of residue adjacent to one another; andfurther optionally e) the molecule comprises two pairs of adjacentcationic amino acids and one or two pairs of adjacent lipophilicresidues, and a pharmaceutically acceptable carrier, or the combined orsequential administration of a pharmaceutical composition consistingessentially of the cytotoxic chemotherapeutic agent and apharmaceutically acceptable carrier, and a pharmaceutical compositionconsisting essentially of the compound and a pharmaceutically acceptablecarrier.
 2. The method of claim 1, wherein the compound is of formula:(I) (SEQ ID No. 1) CCLLCCLLC or (III) (SEQ ID No. 3) CLLCCLLCC;

wherein C represents a cationic amino acid and L represents an aminoacid with a lipophilic R group and in which one of the amino acidshaving a lipophilic R group is a non-genetically coded amino acid, saidcompound optionally in the form of a salt, ester or amide.
 3. The methodof claim 1 wherein each lipophilic R group has at least 9 non-hydrogenatoms.
 4. The method of claim 1 wherein each lipophilic R group has atleast one cyclic group.
 5. The method of claim 1 in which 1 to 3 of theamino acids with lipophilic R groups are tryptophan.
 6. The method ofclaim 1 wherein the compound incorporates a non-genetically coded aminoacid selected from the group consisting of:2-amino-3-(biphenyl-4-yl)propanoic acid (biphenylalanine),2-amino-3,3-diphenylpropanoic acid (diphenylalanine),2-amino-3-(anthracen-9-yl)propanoic acid,2-amino-3-(naphthalen-2-yl)propanoic acid,2-amino-3-(naphthalen-1-yl)propanoic acid,2-amino-3-[1,1′:4′,1″-terphenyl-4-yl]-propionic acid,2-amino-3-(2,5,7-tri-tert-butyl-1H-indol-3-yl)propanoic acid,2-amino-3-[1,1′:3′,1″-terphenyl-4-yl]-propionic acid,2-amino-3-[1,1′:2′,1″-terphenyl-4-yl]-propionic acid,2-amino-3-(4-naphthalen-2-yl-phenyl)-propionic acid,2-amino-3-(4′-butylbiphenyl-4-yl)propanoic acid,2-amino-3-[1,1′:3′,1″-terphenyl-5′-yl]-propionic acid and2-amino-3-(4-(2,2-diphenylethyl)phenyl)propanoic acid.
 7. The method ofclaim 1, wherein the compound has the formula of SEQ ID NO: 23, or asalt, ester or amide thereof.
 8. The method of claim 1, wherein thecytotoxic chemotherapeutic agent is selected from the group consistingof doxorubicin, paclitaxel, cyclophosphamide, gemcitabine and5-fluorouracil.
 9. The method of claim 8, wherein the cytotoxicchemotherapeutic agent is either cyclophosphamide or doxorubicin. 10.The method of claim 1 wherein the compound is a peptide.
 11. The methodof claim 1 wherein the cytotoxic chemotherapeutic agent iscyclophosphamide, wherein the compound has the formula of SEQ ID NO: 23,or a salt, ester or amide thereof, and wherein administration comprisesthe combined or sequential administration of a pharmaceuticalcomposition consisting essentially of cyclophosphamide and apharmaceutically acceptable carrier, and a pharmaceutical compositionconsisting essentially of the compound having the formula of SEQ:ID No.23, or a salt, ester or amide thereof and a pharmaceutically acceptablecarrier.
 12. The method of claim 11, wherein administration comprisessequential administration of a pharmaceutical composition consistingessentially of cyclophosphamide and a pharmaceutically acceptablecarrier, and a pharmaceutical composition consisting essentially of thecompound having the formula of SEQ:ID No. 23, or a salt, ester or amidethereof and a pharmaceutically acceptable carrier.
 13. The method ofclaim 12, wherein the composition consisting essentially ofcyclophosphamide and a pharmaceutically acceptable carrier isadministered prior to the pharmaceutical composition consistingessentially of the compound having the formula of SEQ:ID No. 23, or asalt, ester or amide thereof and a pharmaceutically acceptable carrier.